Guided Tissue and Bone Regeneration Membranes: A Review of Biomaterials and Techniques for Periodontal Treatments

This comprehensive review provides an in-depth analysis of the use of biomaterials in the processes of guided tissue and bone regeneration, and their indispensable role in dental therapeutic interventions. These interventions serve the critical function of restoring both structural integrity and functionality to the dentition that has been lost or damaged. The basis for this review is laid through the exploration of various relevant scientific databases such as Scopus, PubMed, Web of science and MEDLINE. From a meticulous selection, relevant literature was chosen. This review commences by examining the different types of membranes used in guided bone regeneration procedures and the spectrum of biomaterials employed in these operations. It then explores the manufacturing technologies for the scaffold, delving into their significant impact on tissue and bone regenerations. At the core of this review is the method of guided bone regeneration, which is a crucial technique for counteracting bone loss induced by tooth extraction or periodontal disease. The discussion advances by underscoring the latest innovations and strategies in the field of tissue regeneration. One key observation is the critical role that membranes play in guided reconstruction; they serve as a barrier, preventing the entry of non-ossifying cells, thereby promoting the successful growth and regeneration of bone and tissue. By reviewing the existing literature on biomaterials, membranes, and scaffold manufacturing technologies, this paper illustrates the vast potential for innovation and growth within the field of dental therapeutic interventions, particularly in guided tissue and bone regeneration.


Introduction
The field of periodontology has seen considerable advancements in recent years, with a keen focus on therapeutic strategies for restoring periodontal lesions and regenerating lost jawbone through cellular proliferation. Central to this endeavor is the availability of a substantial volume of hard bone tissue, the foundation for successful implant treatments [1].
The complex architecture of a healthy periodontium, with its multilayered structure and dynamic interplay of cells, tissues, and molecular factors, is fundamental to oral health. However, the periodontium can fall prey to a range of pathological conditions, leading to tooth loss and degenerative changes [2]. These conditions necessitate a host of diverse treatment modalities, from traditional periodontal therapies to more contemporary, regenerative procedures.
In the realm of regenerative therapies, guided bone regeneration (GBR) and guided tissue regeneration (GTR) have garnered significant attention. Primarily, GBR focuses on the regeneration of alveolar bone in edentulous regions, while GTR is tasked with repairing compromised periodontal tissues [3,4]. Both these techniques leverage the utility of a porous polymer membrane to physically impede the infiltration of undesirable tissues and cells into the lesion site [4]. This strategy aids in fostering an environment conducive for the proliferation of required cells [5].

Materials and Methods
To conduct this comprehensive review, we sourced primary data from several established scientific databases, including Scopus, PubMed, and MEDLINE. Our objective was to unearth the most relevant and impactful literature relating to the use of biomaterials in guided tissue and bone regeneration procedures.
Our search strategy was developed with a focus on several key terms and phrases pertinent to our study. These include "biocompatible materials", "membrane", "bone regeneration", "tissue regeneration", and "dental biomaterials". We meticulously scanned all abstracts yielded by these search terms and selected full-text articles that aligned most significantly with our study's aims and objectives.
The review process involved a rigorous methodological approach. All selected articles underwent detailed evaluation, where data relating to the membrane types and range of biomaterials used for tissue and bone regeneration were extracted and scrutinized. In addition, we paid particular attention to articles that discussed the role of scaffold manufacturing technologies in these regenerative procedures.
Furthermore, we performed a narrative synthesis of the data obtained. The synthesis was aimed at providing a comprehensive overview of the current understanding and advancements in the use of biomaterials for guided tissue and bone regeneration, as well as the role of membranes in preventing the ingress of non-ossifying cells.
The exclusion criteria applied in the literature review process helped to maintain the focus and relevance of this study. Articles that did not focus on dental tissue and bone regeneration, used non-biocompatible materials, or did not discuss the use of scaffold manufacturing technologies were excluded from the review. Additionally, articles published in languages other than English were not considered.
This methodical approach ensured the selected literature was of high quality and relevant to our study, thereby supporting a more accurate and comprehensive review of the subject matter.

Results
Current periodontal treatment approaches are targeted at minimizing and/or removing inflamed tissues induced by bacterial plaque, repairing deficiencies or structural abnormalities, and regenerating new tissues in the region of lost tissues [11][12][13]. Various methods mentioned previously are only able to stop the progress of the problem, but are unable to reverse the damage or replace the lost tissue [14]. Bone grafting, enamel matrix derivative (EMD) and guided regeneration therapy are now used in the development of tissues that have been infected by periodontal diseases. To a certain degree, the overall structure and function of the damaged tissue can be restored [13].

Historical Viewpoint on Approaches to Periodontal Regeneration
The concept of placing a physical barrier along the tooth root surface after periodontal surgery to prevent epithelial downgrowth was first proposed in the 1970s [15] (Figure 1). methods mentioned previously are only able to stop the progress of the problem, but are unable to reverse the damage or replace the lost tissue [14]. Bone grafting, enamel matrix derivative (EMD) and guided regeneration therapy are now used in the development of tissues that have been infected by periodontal diseases. To a certain degree, the overall structure and function of the damaged tissue can be restored [13].

Historical Viewpoint on Approaches to Periodontal Regeneration
The concept of placing a physical barrier along the tooth root surface after periodontal surgery to prevent epithelial downgrowth was first proposed in the 1970s [15] ( Figure  1).

Figure 1.
Timeline of periodontal regeneration approaches: from the original idea involving a free palatal graft for inhibiting epithelial migration to the most current developments involving additively engineered polymeric multiphasic scaffolds for periodontal tissue engineering.
Indeed, physicians had previously hypothesized that the collapse of gingival tissues into periodontal defects seriously impeded bone resorption [16,17]. Several early experiments suggested the placing of a harvested free palatal graft over the periodontal defect in order to delay or at least obstruct the downgrowth of epithelium around the tooth root surface [15]. Another popular procedure included the insertion of bone grafts (allogenic, autologous, or synthetic) obtained from the patient inside the periodontal defect to regenerate the missing bone [18]. However, neither of these methods is successful for periodontal recovery, and only periodontal healing is observed in the context of a fresh junctional epithelium. After researching the clinical and laboratory evidence, it can be hypothesized that the lack of compartmentalization between the periodontal defect and the underlying soft tissue was the cause of low regeneration rates. The problem of selective periodontal defect repopulation by tissues capable of fostering periodontal regeneration was presented in a series of pioneering papers by Nyman et al., which contributed to the development of the principle of guided tissue regeneration (GTR) [15,19].

Bone Graft Procedure
Bone grafts have been an option for a long time to successfully deal with the effects of periodontal disease, such as bone loss and damage. A bone graft is meant to fill the space that originated from the damaged tissue with a material that possesses certain qualities and characteristics ( Figure 2). There are several types currently available, such as allografts, xenografts, alloplastic, and autograft materials. These kinds of graft materials are able to facilitate natural osseous repair through some mechanisms that have already been properly characterized [18,20,21]: • Osteogenesis: The graft possesses cells that function as seeds for the continuous growth of the tissue by forming a bone matrix. Indeed, physicians had previously hypothesized that the collapse of gingival tissues into periodontal defects seriously impeded bone resorption [16,17]. Several early experiments suggested the placing of a harvested free palatal graft over the periodontal defect in order to delay or at least obstruct the downgrowth of epithelium around the tooth root surface [15]. Another popular procedure included the insertion of bone grafts (allogenic, autologous, or synthetic) obtained from the patient inside the periodontal defect to regenerate the missing bone [18]. However, neither of these methods is successful for periodontal recovery, and only periodontal healing is observed in the context of a fresh junctional epithelium. After researching the clinical and laboratory evidence, it can be hypothesized that the lack of compartmentalization between the periodontal defect and the underlying soft tissue was the cause of low regeneration rates. The problem of selective periodontal defect repopulation by tissues capable of fostering periodontal regeneration was presented in a series of pioneering papers by Nyman et al., which contributed to the development of the principle of guided tissue regeneration (GTR) [15,19].

Bone Graft Procedure
Bone grafts have been an option for a long time to successfully deal with the effects of periodontal disease, such as bone loss and damage. A bone graft is meant to fill the space that originated from the damaged tissue with a material that possesses certain qualities and characteristics ( Figure 2). There are several types currently available, such as allografts, xenografts, alloplastic, and autograft materials. These kinds of graft materials are able to facilitate natural osseous repair through some mechanisms that have already been properly characterized [18,20,21]   Diverse graft materials can be categorized into four general types that are shown in Table 1. In the past, research publications have reported that at least 3.0 mm of bone height can be acquired, regardless of which material the graft is made of [21,22]. Probably the best option for osteogenesis, osteoconduction, and osteoinduction is autologous bone Diverse graft materials can be categorized into four general types that are shown in Table 1. In the past, research publications have reported that at least 3.0 mm of bone height can be acquired, regardless of which material the graft is made of [21,22]. Probably the best option for osteogenesis, osteoconduction, and osteoinduction is autologous bone since it shares all of its properties with the surrounding bone. In this type, a part of the bone structure is extracted from a normal and un-damaged area of the patient who is getting the graft. The structure that is compatible and inherent to this type makes it very advantageous since it has the same vital bone structures in critical regions and includes nutrients, proteins, and cells as those found in the affected site. However, the autoimmune grafts come with some disadvantages, such as increasing the patient's pain at the same site of the excision. In addition, only small amounts of bone can be extracted without incurring permanent damage to the patient.
Due to these drawbacks, several xenografts, bone grafts, as well as allogeneic materials have been designed and approved for commercial use [23]. It is important to note that clinicians must take into account the risks associated with these materials, such as infection, resorption, and immune responses. Currently, two types of allografts are available: freezedried bone allograft (FDBA) and demineralized freeze-dried bone allograft (DFDBA). These grafts are pre-treated chemically; they keep their osteoinductive capabilities due to the conservation of certain proteins, such as BMPs and TGF-s. These proteins work as powerful growth stimulants and induce the mobilization of cells from the mesenchyme into the implant. The overall process of demineralization enhances the excretion of these highly important factors into the extracellular medium [21,24].
The grafts that are extracted from animals, mainly farm cattle, are called xenografts. They go through chemical treatment in order to remove their inherent antigens, in order to avoid the human body's natural immune response. The greatest advantage in using these products lies in the fact that the general structure of the graft can be maintained, and this is because of the prolonged amounts of time required for these materials to be resorbed. These materials are capable of osteoconductivity and little else. However, the overall safeness and efficacy of them have been proved to be clinically relevant [21,25].
Due to the risks related to these types of grafts, scientists have also conducted investigations into the use of synthetic alternatives, which include composite grafts, polymer and inorganic materials, to repair osseous tissue [26]. In particular, materials such as hydroxyapatite and β-tricalcium phosphate (β-TCP) have become of increasing clinical importance when studying periodontal diseases. Furthermore, even though synthetic materials lack osteogenic and osteoconductive capabilities, their osteoconductive properties make them excellent options for bone regeneration. Recently, in a study by Schmidlin et al. (2013), it was found that polylactide-coated TCP was sufficient to repair problems in the rabbit's bone structure, all of this while retaining its biocompatibility [27].
Certain products of natural origin, such as coral, have been shown to be able to be used as bone grafts due to their similarity to human bone structure [28]. Similarly, to synthetic materials, numerous natural products have been proven safe for human use and are very cost-efficient. However, they are still only osteoconductive [21].

Guided Regeneration Therapy
The key aim of periodontal regeneration is to establish new cementum with periodontal ligament (PDL) fiber attached to the alveolar bone and promote new bone growth. Currently, there are two surgical approaches that have been used for regenerating periodontal tissues. These are guided tissue regeneration (GTR) and guided bone regeneration (GBR) [12,29,30]. The concept of guided tissue/bone regeneration began in the late 80s and was developed by Nyman et al. (1990) based on Melcher's theory. This theory hypotheses that when cells with regenerative capabilities are associated with damaged tissues, they can actually be used to aid in the regeneration of that tissue [19]. The hypothesis has been successfully proven in various animal experiments, and the principle of guided tissue regeneration (GTR) has been confirmed [12,15,29].
GTR refers to the procedure of regenerating periodontal tissue through the use of an occlusive barrier membrane between gingival (epithelial) and alveolar bone/PDL tissue. In this operation, an occlusive membrane will be inserted onto the surgical site in order to inhibit the migration of connective and epithelial tissue through the surgical site [12,15,29]. Progenitor cells that existed in the lining of the residual periodontal ligament, corresponding alveolar bone, or blood can then re-colonize the root region and divide into new periodontal supporting components [29]. The guided bone regeneration (GBR) approach is often used to repair defective alveolar ridges before or in conjunction with the placement of a dental implant at extraction sites. In GBR, a bone defect is protected by a membrane to stop fiber tissue intrusion into the site of the graft and to promote the development of a new bone. Intrabony abnormalities and furcations are also treated with GBR [12,15,29].
In order to promote the development of healthy bone structures surrounding bone defects, GBR permeable membranes can be utilized ( Figure 3). GBR membranes can also be used in order to preserve the socket area that may be around the tooth due to the presence of periodontal disease, and can even be used to regenerate bone structure at the tooth site after it has been misplaced or extracted [31]. [12,15,29]. Progenitor cells that existed in the lining of the residual periodontal ligament, corresponding alveolar bone, or blood can then re-colonize the root region and divide into new periodontal supporting components [29]. The guided bone regeneration (GBR) approach is often used to repair defective alveolar ridges before or in conjunction with the placement of a dental implant at extraction sites. In GBR, a bone defect is protected by a membrane to stop fiber tissue intrusion into the site of the graft and to promote the development of a new bone. Intrabony abnormalities and furcations are also treated with GBR [12,15,29].
In order to promote the development of healthy bone structures surrounding bone defects, GBR permeable membranes can be utilized ( Figure 3). GBR membranes can also be used in order to preserve the socket area that may be around the tooth due to the presence of periodontal disease, and can even be used to regenerate bone structure at the tooth site after it has been misplaced or extracted [31]. The efficacy of this therapeutic approach was later verified by Gottlow et al., who effectively extended it to a large group of patients [32,33]. Subsequently, the theoretical and biological basis of GBR has been confirmed during the last three decades in several studies [34], and the effectiveness of the procedure has been shown in a multitude of clinical trials [35] and recorded in comprehensive reviews [15,36].
Guided regeneration has been shown to have many benefits when it comes to tissue regeneration over more conventional surgery approaches such as open-flap debridement, which is usually used to address intrabony defects and mild to moderate furcations [15,36].
The concept behind bone augmentation protocols mainly relates to the enhancement in function and aesthetics and can also be used to improve the functions of fixed dental prosthetics, including dental implants and fixed dentures. This procedure greatly helps in correcting contour deficiencies associated with the replacement of artificial teeth as well as offering a friendlier solution to altered speech patterns due to the uncovered areas frequently left between gingival tissues and the restoration [15,37].

Biologic Principles for Guided Regeneration Therapy
In order to successfully develop an engineered tissue, the following essential elements are required: properly defined levels and patterns of regulatory signals; an The efficacy of this therapeutic approach was later verified by Gottlow et al., who effectively extended it to a large group of patients [32,33]. Subsequently, the theoretical and biological basis of GBR has been confirmed during the last three decades in several studies [34], and the effectiveness of the procedure has been shown in a multitude of clinical trials [35] and recorded in comprehensive reviews [15,36].
Guided regeneration has been shown to have many benefits when it comes to tissue regeneration over more conventional surgery approaches such as open-flap debridement, which is usually used to address intrabony defects and mild to moderate furcations [15,36].
The concept behind bone augmentation protocols mainly relates to the enhancement in function and aesthetics and can also be used to improve the functions of fixed dental prosthetics, including dental implants and fixed dentures. This procedure greatly helps in correcting contour deficiencies associated with the replacement of artificial teeth as well as offering a friendlier solution to altered speech patterns due to the uncovered areas frequently left between gingival tissues and the restoration [15,37].

Biologic Principles for Guided Regeneration Therapy
In order to successfully develop an engineered tissue, the following essential elements are required: properly defined levels and patterns of regulatory signals; an abundance of progenitor cells; a sufficient blood supply; and an appropriate biomaterial scaffold [38]. Whereas cells serve as the processing facility for the formation of newly formed tissue and the differentiation of cells. Cells require external stimuli in order to stimulate growth and matrix synthesis. These can be provided by growth factors or morphogens. New vascular networks are promoted as a result of angiogenic signals, which supply nutrients for tissue growth and maintenance. The three-dimensional architecture of scaffolds aids in directing cell regeneration [11,15]. The healing process for periodontal surgery wounds follows the same three stages as any other incisional wound. Initially, a fibrin clot is shaped along the flap's margin and the root surface. Then, a connective tissue matrix attached to the root surface takes the place of the fibrin clot [39]. By keeping the fibrin attached, a new connective tissue bond can form on the root's outer surface. However, a long-bonding epithelial connection forms if the limit of the fibrin clot's tensile strength is surpassed [15,40,41].
In general, the method of periodontal healing is more complicated than other wounds by considerations such as the involvement of various specific cell types and complexity of attachments; avascular root surfaces; different microbial flora; and stromal-cellular interfaces [42]. The first biological reaction that happens after the installation of the barrier membrane is the action of the tissue-membrane interface to absorb the plasma protein.
Hence, the related growth factors and progenitor cells, which play an important role in tissue repair, are attracted to the surface of the membrane with the help of proteins [43]. In order to provide nourishment to the new tissue in the barrier membrane, which protects defects, much of the vascular supply comes from blood vessels that originate in the marrow [44]. This further explains why it is vital to plan multiple perforations in cortical bone (also called intra-marrow penetration), as this assists in the production of an excess of angiogenic and osteogenic cells as a means of creating new blood vessels and constructing new bone tissue. This serves two objectives: first, it induces bleeding or blood clotting around the grafts in order to induce bone formation around the grafts, and second, it increases the number of factors that raise the likelihood of bone growth [15,37,41].
There is also controversy about the effector cells in periodontal regeneration. Some reports indicate that PDL cells have the potential to behave as osteoblasts or cementoblasts when they are supplied with growth factors and allowed to proliferate. Other data indicate that PDL cells have the ability to regulate mineral formation, so this will help avoid ankylosis when undergoing regeneration [45]. In other studies, PDL cells in vivo and in vitro have been reported to exhibit minimal osteoblastic properties [42,45]. However, other studies argue that osteoblasts, and not PDL cells, are responsible for generating cementum-like material [17,46,47]. Such variations can be attributable to PDL cell heterogeneity, differing study designs, and/or loss of cell properties defined in vitro research. In summary, the majority of evidence points to PDL cells as the main source. Some reports also point to bone cells as the origin of regenerative cells [15,41,48].

Requirements of GTR/GBR Membranes
GTR and GBR membranes need to fulfil specific requirements to be most effective and successful [49]. These requirements can be summarized as follows:

•
Cell exclusion: A growth guide membrane can be used to separate several types of unwanted tissue (e.g., epithelial cells) as well as get access to the site of interest [50]. • Framework: A more rigid framework is frequently necessary when clinical cases need more space maintenance in order to prevent membrane compression into the defect site. Bone grafts can provide this support [49,51]. • Porosity: In order to achieve appropriate cell growth and proliferate, the cells must have an underlying high-pore structure [9]. • Degradation: It is important to provide a degradation profile that suits the tissue regeneration, which takes approximately four to six weeks. Ideally, the membrane should fully degrade after it fulfils its purpose without leaving any residual materials [52]. • Stabilization: To prevent mechanical disturbances from the outside and overhanging of flap movement during the process of healing. Mini screws or sutures, can be used to keep the membrane in place [49].

•
Clinical manageability: The membrane and the barrier need to possess physical characteristics that enable their handling by the clinician [53]. • Biocompatibility: Inflammation should be avoided at all costs in order to avoid increased morbidity and costs [49,54]. Owing to the large number of scaffolds that can be made from a wide range of materials, these materials have differing degrees of degradation and integrity. These scaffolds can often induce immune reactions in the hosts [55]. The following section discusses commercial membranes, whether non-absorbable or absorbable, in guided regeneration therapy.

Types of Commercial Membranes Used in Guided Regeneration Therapy
Barriers used in bone/tissue regeneration procedures have varying degrees of deterioration and properties and, therefore, have generally been divided into resorbable or non-absorbable membranes. Gottlow, (1993) was the first to divide these membranes into two generations depending on when they were created and developed; the first generation consists of non-resorbable membranes, while the second generation contains all resorbable membranes [56]. Before Elgali et al. (2017) reviewed this classification and added a new group, Third generation, its membranes rely on naturally derived sources combined with bone grafts and alternative materials to provide structural support to the defect site and to promote the intrinsic regenerative potential of the host tissue [5,15,41].
The following sections discuss the various commercially available periodontal membranes classified as non-resorbable or resorbable materials and are summarized in Table 2. Table 3 includes a list of the primary biomaterials used in bone tissue engineering, along with their key characteristics.

First-Generation Membranes: Non-Resorbable Guided Membranes
In the 1960s and 1970s, the first generation of barrier membranes were developed with the goal of achieving a sufficient mix of physical qualities that would match those of the replaced tissue while also eliciting a low toxic response in the host [15,41].
In the initial GTR experiments, an occlusive membrane consisting of a bacterial filter made from cellulose acetate (Millipore, Burlington, MA, USA) was utilized. These experiments were conducted by Nyman et al. in 1982 [57]. Due to its toxicity, this form of membrane was not appropriate for clinical applications despite serving its goal. In later trials conducted in the 1990s, membranes of expanded polytetrafluoroethylene (e-PTFE) created specifically for periodontal regeneration were applied (Gore Tex Periodontal Material) [5,15,58].
E-PTFE has a dual-layered structure with pores measuring 5-20 microns in diameter. One side of this membrane is 1 mm thick and has an open microstructure that is 90% porous, preventing epithelial penetration; the other side is 0.15 mm thick and has a porous structure that is 30% thick, allowing space for new bone production [59]. Several investigations have shown that e-PTFE is effective, as described by Liu, J. and Kerns, D.G. [59] However, due to their very porous structural design, they have a high rate of exposure, which is seen as a major disadvantage, in addition to the need for additional surgery to remove them from the location of the newly created tissue.
A high-density d-PTFE membrane with hole sizes of less than 0.3 microns was created to counteract the drawbacks of e-PTFE [60]. In spite of the advantage of non-sticking of tissues to the membrane, which made its removal easy and simple, in addition to its ability to properly regenerate the bones even in exposed cases due to its modified transparency, However, the d-PTFE has limited flexibility, causing it to collapse into the site of the defect [59].
Titanium-reinforced e-PTFE and d-PTFE membranes were produced in order to address the lack of mechanical stiffness that appeared in the initial e-PTFE and d-PTFE membranes [61,62]. However, the requirement for a second surgery to remove the membrane is the most significant disadvantage, similar to other non-resorbable membranes, as well as the rigidity of titanium mesh can create some difficulties during removal due to the need for orthopedic fixation devices such as orthopedic screws. Ti-mesh also appears frequently, which restricts its applications, particularly in aesthetic applications. [63].

Second-Generation Membranes: Resorbable Guided Membranes
Regarding the several applications of GTR and GBR, an absorbable membrane has been proposed as a replacement to the membrane discussed in the previous section in order to minimize its limitations, most notably the requirement for extra surgery to remove the membrane. Based on the origin of the material used to produce the membrane, absorbable membranes are classified into two main groups: natural membranes and synthetic membranes [5,15].

Natural Resorbable Membranes
Numerous natural polymers have been shown to be useful in tissue engineering, which include polysaccharides (cellulose, alginate, starch, hyaluronic acid derivatives, chitin/chitosan), and proteins (soy, fibrin gels, collagen, silk) [26,64]. Natural polymers are also strongly coordinated and may include extracellular substances known as ligands that are essential for binding with cell receptors that can support cell adhesion and function. However, on either side, their medicinal use is constrained by their shortage and the complexity of their processing into scaffolds. In addition, they can induce an immune reaction since natural polymers can lead to cells growing at different developmental stages. Moreover, the rate of degradation varies between patients due to the enzymatic processes involved [5,15,65].
Collagen and chitosan appear to be the two main components of most natural membranes, which are naturally derived from many animal sources. Perhaps the most notable one is the use of bovine Achilles tendon (Cytoplast ® ), human skin (Alloderm ® ), or porcine skin (Bio-Gide ® ) to produce tissue-derived membranes based on collagen [66,67].
The presence of collagen in these membranes is a significant biological feature, as it contributes to many biological activities. Besides being biocompatible, biodegradable, and hemostatic, it also helps in attracting the gingival fibroblast and periodontal ligament (PDL) in addition to augmentation of the soft tissue. Using collagen type I, most of the commercially available collagen membranes are produced and developed, as well as a mixture of collagen types I and III [59]. In vivo experiments found that the collagen-dependent membrane showed some drawbacks, such as its modest efficiency, especially during degradation. Moreover, it may cause ethical and religious issues as well as be a cause of disease transmission [30]. Many biophysical characteristics and collagen framework stabilization can be improved by a number of methods that depend mainly on mechanical and chemical crosslinking, such as adding substances such as glutaraldehyde (GA), diphenyl-phosphoryl azide (DPPA), hexamethylene diisocyanate (HMDIC), and formaldehyde (FA), genipin (Gp), in addition to using ultraviolet light and irradiation [15,68,69].
Collagen structural integrity and mechanical properties are affected by the rehydration protocol, i.e., inserting a cross-linking agent that is natural, genipin into the AlloDerm ® [68]. Studies have shown that extending the exposure time for genipin (Gp) to 6 h from 30 min significantly improves tensile strength in comparison with controls. Additionally, according to other studies, cross-linking is effective for controlling prolonged biological degradation, decreasing tissue amalgamation, and vascular depression, as well as for decreasing epithelial migration [70]. A biocompatible reaction of the membrane made of silk fibroin by osteoblast was also observed, which could be used for GBR as an alternate barrier membrane [71].

Synthetic Resorbable Membranes
Synthetic polymers have several advantages over natural polymers, including the ability to have their properties tuned, an infinite variety of forms, and well-established structures. The support that is provided by synthetic biomaterials can make it possible to restore the structural integrity and functional capacity of diseased or damaged tissues [72]. Synthetic polymers can be modified in terms of their molecular weight, molecular structure, and physical and chemical properties simply unlike polymers derived from natural sources, through the addition of certain functional groups and side chains, synthetic polymers may be self-cross-linked or cross-linked with enzymes or other bioactive molecules [73][74][75].
Synthetic biomaterials have the limitation of lacking cell attachment sites and requiring chemical alterations to improve cell adherence [76]. Physicochemical and mechanical properties of several commercially available synthetic polymers are close to those of biological tissues [77]. The mechanical and physical properties, such as stiffness, Elastic modulus, and degradation rate, are repeatable and predictable throughout a wide spectrum [5,15,76].
The most commonly investigated synthetic degradable materials are poly (-hydroxy esters), which include PCL, PGA, PLA, and their copolymer PLGA, and poly(ethers), which include PEO and PEG, PVA, and PU. These are perhaps the most common examples, however there are now many other synthetic materials being studied [72,76,77]. These polymers all have varying degrees of biodegradability, biocompatibility, and mechanical qualities; nevertheless, there is not a single polymer that possesses all three of these essential properties at the optimal amount [78].

PGA-Based Membranes
Polyglycolic acid is organically created through polycondensation of glycolic acid or ring-opening polymerization (ROP) of glycolide. PGA has a very high fusion point at around 226 • . PGA can be processed through hydrolysis, and its by-products can be processed by the Krebs cycle and then eliminated. It is generally used as a suture, but it can also be used as a PLA co-polymer [79]. Resolut ® is another commercially available product consisting of two layers: a PLGA compact layer that prevents epithelial cell penetration, and a porous network of polyglycolide fibers that promotes tissue integration. Histological studies showed similar effectiveness to non-resorbable membranes and complete resorption 5-6 months after placement [80,81].
Fibers of polyglactin 910, a copolymer of glycolide and L-lactide (9:1 wt/wt), were used to produce a woven mesh (Vicryl Periodontal Mesh ® ). The polyglactin 910 is inert (no reactions in the surrounding tissue during its adsorption were observed), not antigenic, and preserves its physicomechanical properties during the first 3-4 weeks [82]. Although animal studies indicated a lack of tissue integration and recession formation, clinical evaluation suggested a similar effectiveness as compared to that of other GBR membranes [56,80,81,83].

PCL-Based Membranes
Poly-ε-caprolactone is a polymer with some crystal-like properties that melts at approximately 60 • C. It possesses a relatively slow degradation rate, which makes it better suited for long-term applications such as drug delivery systems. A plethora of studies have evaluated this approach and determined that PCL is an effective delivery polymer. Additionally, its physical properties can be modified through the addition of materials such as PGA or PLA. It also possesses applications in osseous scaffolding [84][85][86]. Membranes based on copolymers of lactic acid and e-caprolactone have been produced, showing a lower degradation time as compared to pure PLA membranes. PCL is characterized by higher hydrophobicity and lower water solubility than PLA, PGA and their copolymers. A commercial product, called Vivosorb ® , consisting of poly(DL-lactide-ecaprolactone), was found to be biocompatible, non-cytotoxic, occlusive and space maintaining [87].

PLA-Based Membranes
Polylactic acid is synthesized similarly to PGA, through ring-opening polymerization of its lactic acid (HOCHCH3COOH) [88]. Its structure can be seen in Figure 4. Membranes based on copolymers of lactic acid and e-caprolactone have been produced, showing a lower degradation time as compared to pure PLA membranes. PCL is characterized by higher hydrophobicity and lower water solubility than PLA, PGA and their copolymers. A commercial product, called Vivosorb ® , consisting of poly(DL-lactideecaprolactone), was found to be biocompatible, non-cytotoxic, occlusive and space maintaining [87].

PLA-Based Membranes
Polylactic acid is synthesized similarly to PGA, through ring-opening polymerization of its lactic acid (HOCHCH3COOH) [88]. Its structure can be seen in Figure 4. PLA is one of the best biopolymers due to its biocompatibility and ease of biological degradation. Because of its properties, it has been used in various biomedical and clinical applications [90][91][92]. PLA exists in three optical isomers, specifically in its L-lactide form  [88,89].
PLA is one of the best biopolymers due to its biocompatibility and ease of biological degradation. Because of its properties, it has been used in various biomedical and clinical applications [90][91][92]. PLA exists in three optical isomers, specifically in its L-lactide form as (PLLA) and its D-lactide form as (PDLA). Additionally, it has a hybrid form (PDLLA) [93]. Because of its nature as an amorphous crystal, PDLLA degrades quicker than other forms of PLLA, in less than half a year [94,95].
The Guidor ® Matrix Barrier is a bioresorbable membrane, first used for the regeneration of tissues in periodontology, consisting of polylactic acid treated with acetyltributylcitrate to achieve flexibility to guarantee close barrier adaptation to the bone defect. The Guidor ® Matrix Barrier has a matrix with two differently perforated layers. The external layer, allowing integration of the overlying gingival flap, presents large pores (rectangular shape) to promote tissue integration and to enable gingival connective tissue to penetrate quickly into the matrix. The inner layer presents small pores (circular shape), able to retard tissue penetration while allowing nutrient permeation. The two layers are separated by many inner spacers, forming an interspace into which tissue can grow. According to the manufacturer, the barrier structure is not affected by the material degradation for at least the first 6 weeks, and a complete resorption takes place after one year due to hydrolysis [56,83].
Atrisorb ® membrane is the first liquid product adapted directly at the surgical site: it consists of poly-DL-lactide acid dissolved in N-methyl-2-pyrrolidone. An irregular membrane is produced after polymer exposure to 0.9% saline solution for 4-6 min in a special cassette, in which it is possible to cut it into the desired shape. Membrane thickness is 600-750 µm, and it is positioned into the defect site by applying a moderate pressure. A histological complete resorption was observed 6-12 months after implantation [96]. Clinical studies reported its efficacy in the treatment of periodontal defects [97].
The Epi-Guide ® Bioresorbable Barrier Matrix is a porous membrane consisting of D-L polylactic acid with a unique three-layer technology, used as an adjunct to periodontal restorative surgery. The Epi-Guide maintains its structure and functions for 5 months after implantation, with a complete bioresorption after one year [98]. The layer in contact with the gingiva is porous to promote fibroblast infiltration and attachment. On the contrary, the layer in contact with bone defects has a limited porosity that supports fluid uptake, helps adherence to the tooth surface, and inhibits fibroblast movement [98,99].
For successful periodontal tissue regeneration, the materials used must be compatible with living tissue and favorable in terms of mechanical properties. These specifications cannot be fulfilled by conventional single-component polymer materials. As a result, designing and preparing multicomponent polymer structures represents a promising approach for developing multifunctional biomaterials [100].

Third-Generation Membranes
By reviewing the previous absorbable and non-absorbable membranes, interests should arise in developing a new membrane which has a more advanced role as a barrier membrane and has an additional function such as releasing beneficial agents such as bioceramic, antibiotics, growth factors, and adhesion factors into the wound. The substancereleasing membrane should have a proper release time according to the environment of the graft site [9].

Resorbable Membranes Based on Polymer Composites
Polymer Blends Polymer membranes must meet a few key criteria for successful guided bone and tissue regeneration (GBR and GTR, respectively), appropriated mechanical and physical properties, a suitable degradation profile, as well as the necessary strength to provide an effective barrier function and resist decomposition [101]. Due to a variety of requirements, a single polymer fails to meet all critical criteria. For instance, naturally occurring polymers cannot provide the required mechanical strength and suitable degradation profiles, while synthetic polymers are unable to interact with biological tissues. On the other hand, polyester membranes turn rigid and brittle after introduction to phosphate buffered saline or artificial saliva solution [102].
Therefore, the issue of developing membranes with the necessary mechanical properties, the expected rate of decomposition, as well as a structure similar to the natural extracellular matrix (ECM) remains topical [103]. A potential solution is to combine two or more polymers in order to offset their disadvantages and find a mutually reinforcing effect.

Natural Polymer and Synthetic Polymer Blends
Natural polymers are known for their increased biocompatibility and bioactive properties compared to synthetic counterparts. For instance, gelatin shows multiple integrinbinding sites to promote cellular adhesion and differentiation [104,105]. Mixing polymers of natural and synthetic origin should provide opportunities for taking advantage of both of them. For example, a material based on an amalgamation of gelatin with PCL has excellent biocompatibility as well as the essential mechanical, physical, and chemical qualities. Its unique properties allow it to be used in cartilage tissue engineering [106,107], neural tissue engineering [108], as well as GBR and GTR [104,105,109]. That being said, chemical segregation between PCL molecules and gelatin is a factor inhibiting the development of composites with the required characteristics.
It has been found that acetic acid can favorably affect the rate and strength of miscibility between PCL and gelatin. For this reason, it is effective for implementation when homogeneous nanofibers with improved performance are required [110,111]. The biodegradation period of such membranes is also appropriate for tissue regeneration [110].
The PLLA/chitosan multilayer membrane proposed by Ku et al. (2009) has shown excellent potential for utilization in GBR and GTR [112]. The membrane has external chitosan netting that promotes the adhesion of cells from nanoporous PLLA located in the middle layer. This layered structure allows for improved mechanical strength and integrity preservation for up to eight weeks.

Natural Polymers Blends:
Despite its natural origin, the bioactivity and mechanical properties of chitosan are inferior to those of protein polymers. In order to improve its properties, chitosan is often blended with other polymers. Due to the presence of free carboxyl groups in the structure of gelatin, it successfully blends with chitosan and forms a stable hydrogen bond with it. The ability of gelatin/chitosan membranes to maintain cellular adhesion and proliferation is better than that of gelatin and chitosan on their own [113]. Moreover, the enhancement by proanthocyanidin gives the gelatin/chitosan bond greater stability and improves its mechanical properties compared to membranes constructed from gelatin or chitosan and gelatin blend [113]. An example of the successful integration of natural polymers is a three-layer membrane with a chitosan interlayer sandwiched between two collagen membranes featuring 20 wt % HA [114]. Hunter and Ma, (2013) have shown that membranes based on hydroxyapatite/chitosan/gelatin can promote the growth of bone marrow mesenchymal stem cells (hBMSC) whilst improving the pace of osteogenic differentiation [115]. Research data assure that gelatin/chitosan or collagen/chitosan membranes possess adequate mechanical and structural properties to be implemented as a barrier membrane. Therefore, they demonstrate the potential to be used in bone and tissue regeneration.

Synthetic Polymer Blends
PLA, PLGA, PCL, and some other aliphatic polyesters are essential components for the production of fibrous scaffolds required for drug delivery systems and tissue regeneration [116]. At the same time, PLGA is characterized by reduced mechanical strength, which makes it impossible to maintain the scaffolding structure during in vitro and in vivo clinical trials. When PLGA was reinforced with other polymers such as PCL, applied in an equal ratio, the compressive strength of the PCL/PLGA scaffolding was far superior to the strength ensured by PLGA alone [117]. Cytological investigations have demonstrated that penetration of human embryonic kidney 293T cells can be prevented by using PDLLA/PLGA electrospinning devices with an appropriate degradation rate and effective cell occlusion for the purpose of GTR. In addition, implantation of a subcutaneous implant in rats demonstrated that PDLLA/PLGA membranes with a composite ratio of 70/30 and 50/50 are able to double as a physical barrier that stops cellular infiltration for a duration of 13 weeks [103]. These data suggest that PDLLA/PLGA membranes can become an effective barrier membrane for tissue regeneration purposes [103]. Along with this, composite membranes fabricated from PLA/PCL, PLGA/PCL, and other synthetic compounds may be deemed as a promising technology for GBR and GTR [117][118][119].

Floreon™ blend
Floreon is a new sustainable polymer blend created by Floreon-Transforming Packaging Limited in collaboration with the University of Sheffield and certified by the EN13432 standard [120,121]. Based on PLA, Floreon is composed of renewable components, which is likely to improve its mechanical and chemical properties [120,121].
In comparison to pure PLA, Floreon exhibits a remarkable four-fold increase in strength and is less susceptible to cracking and breakage during the manufacturing process and testing phases, as demonstrated by Floreon 3D (2014) and Floreon (2018). The compound has a maximum tensile strength of approximately 1.6 GPa while the elongation at break (fracture strain) is 14% [120][121][122]. Moreover, in comparison to PLA, Floreon exhibits enhanced thermal performance. It has a melting point of 210 • C [120,121,123], a crystallization temperature of 85 • C [123], and a glass transition temperature of 65 • C. Floreon is extruded at temperatures between 170 and 180 • C. However, since its destruction threshold is 250 • C [120,123], technological processes should not exceed 220 • C. In order to prevent moisture absorption, the material is dried at 65-90 • C after crystallization [120]. Floreon may undergo thermoforming, compounding, and injection molding processes in addition to extrusion (including film extrusion).
There are currently eight Floreon variants labelled in the range FL100-FL800 [120,121]. Due to its resilience to ultraviolet radiation, the Floreon blend is more effective than PLA for 3D printing and lithographic printing [120,121].
Although the Floreon was originally designed for the packaging industry, it has recently been investigated as a scaffold for musculoskeletal applications. The conclusion drawn is that the Floreon blend showed great promise for use in bone tissue regeneration [124].

Bio-Ceramic/Polymer Composites
The incorporation of polymer composites, bioceramic components, and the structural mimicry of bone extracellular matrix (ECM) can be advantageous for the development of biomaterials that are used for guided bone regeneration (GBR) and guided tissue regeneration (GTR) [9]. Hydroxyapatite (HA) [125], carbonated hydroxyapatite (CHA) [126], bioactive glass (BG) [127], βtricalcium phosphate (β-TCP) and other bioceramics have been widely used in bone tissue engineering and shown to have excellent biocompatibility and osteoconduction properties [128,129].
The use of bioactive ceramics in GTR and GBR has a positive impact on mineralization and cell activity boost on polymer membranes, which suggests the required osteoconductivity and osteoinductivity [127,[130][131][132][133]. On top of that, bioactive compounds are capable of affecting mechanical properties in a beneficial way [134]. While pure PLGA has a tensile strength of 0.49 MPa, the inclusion of 10-30 wt % nanoapatite into a membrane helps lift it to 0.61 MPa [135]. At the same time, the introduction of bioceramics is able to neutralize the acidic derivatives of PLA, chitosan, and other polymers formed due to their decomposition in an alkaline medium [130,136,137]. According to Khan et al. (2008), composite membranes have the ability to effectively and biomimicking preserve the structural and biological functions of damaged dense tissues [138].
Because hydroxyapatite is osteoinductive, it accelerates bone regeneration and allows the bio-ceramics component to connect directly to the regenerated bone, bypassing connective tissue. The composite has found wide application in orthopedic surgery and dentistry dealing with hard tissue restoration [26,139,140]. Inorganic-organic composites that emulate the structure of human bone offer increased toughness inherent in polymeric materials and the compressive strength characteristic of inorganic components. Their beneficial nature makes it possible to create bioactive materials with improved mechanical properties and degradation profiles. Such composites are stable enough since the alkalinity of the inorganic fraction (for example, hydroxyapatite) balances the acidic substances formed during the autocatalytic decomposition of polymers (such as PLA) [141]. Fabricated PCL/nHA nanocomposites possess properties characteristic of HA ceramics and simultaneously provide the qualities of synthetic polymer PCL, namely, osteoconductivity and biocompatibility [26,142]. Studies of poly (lactic acid) (PLA) nanofibers containing hydroxyapatite filler showed that HA contributes to the improvement of the mechanical and thermal features of the nanofibers [143]. In addition, testing of the β-chitin-HA composite membrane made it possible to detect inclusions of apatite on the surface of β-chitin membranes. This finding indicates increased biocompatibility and provides a suitable foundation for successful cell attachment, adhesion, and proliferation [144].
Bioactive glasses (BSs) are osteoconductive and osteoinductive silica biomaterials with a SiO 2 -CaO-P 2 O 5 structural grid. The introduction of BG stimulates osteogenesis and angiogenesis both in vitro and in vivo [145,146], and also generates high-performance collagen composites in imitation of bone mineralization. In particular, it is involved in the release of Ca, P, and Si and the subsequent deposition of Ca and P as well as amorphous Ca-P crystals on the implant surface. The following chemical dehydration reactions convert these crystals to hydroxycarbonate apatite (HCA) [147]. In a similar way, wollastonite (CaSiO 3 ) gives up Si and Ca ions, which induce the acceleration of osteogenic differentiation and cell multiplication. Simultaneously, this can lead to deposits of bone-like apatite on the surface of the implant after it has been introduced to simulated body fluids (SBF) [148,149]. Wollastonite exhibits the capability of increased structural mechanical strength, angiogenesis, and bone regenerative capacity. Despite this, it should be subject to further research to identify the bioactivity, osteogenic capacity, and immunogenicity of polymer composites when implanted in humans. These studies are driving the development of polymer/bioceramic based composites offering the advantages of both components [124].    Unknown mechanism of cellular proliferation and differentiation [50] Multiphasic Scaffolds of Periodontal Tissues Regeneration A multiphasic scaffold is defined by the differences in its architecture (porosity, pore organization, etc.) and its chemical composition, which usually mimics to some degree the structure or cellular and biochemical composition of the native tissue. Multiphasic scaffolds are designed to impart biomimetic functionality to tissue-engineered bone and soft tissue grafts have been recognized for some time as having the potential to facilitate clinical translation in the field of orthopedic tissue engineering, and more recently in the field of periodontal tissue regeneration [181].
In recent years, guided tissue regeneration and guided bone regeneration (GTR and GBR) approaches have been widely used to manage periodontitis. These membranes have separate functions on each side. The occlusive periodontal membrane acts as a barrier to inhibit the ingrowth of epithelial and undesirable tissues into the defective area during periodontal wound healing, whereas the opposite side promotes regeneration of periodontal tissues [181,182]. GTR/GBR membranes must have certain features, particularly those utilized in large-area repair, such as mechanical stability, osteoconductivity, and a balance between membrane degradation and tissue regeneration, all of which are required for the membranes to function [181]. In a number of studies, bilayer GTR/GBR membranes have been utilized as a treatment for periodontal diseases; here are a few examples from the last few years.
The Yoshimoto group has recently developed bilayer membranes based on PLGA or PCL [183,184]. These membranes consisted of a solid layer and a porous layer that, respectively, served as a barrier and provided cell support. By changing the freeze-drying temperature, they were able to control the thickness of each layer. These membranes were found to be more functional than monolayer membranes, with evidence suggesting that their porous structure aided in the osteogenic differentiation and proliferation of mesenchymal stem cells. In vivo studies also demonstrated that the PLGA bilayer membrane promoted bone regeneration with significantly increased bone formation compared to that with a monolayer membrane [184]. Requicha et al. (2016), in a related method, created a biphasic scaffold made of a porous fibrous PCL/starch scaffold for enabling bone ingrowth and an occlusive membrane developed using the same matter [185]. In this technique also, the occlusive membrane was devised to sustain periodontal ligament regeneration by inhibiting epithelial and gingival tissue invasion of the periodontal defect, hence carefully choosing osteoblast and periodontal fibroblast ingrowth as per the GTR law. After performing in vitro analyses, Requicha et al. (2014) and Requicha et al. (2016) discovered a high potential for osteogenesis, which is a key aspect of periodontal regeneration [185,186]. Park et al. (2010) suggested an approach that involves computer-assisted design and manufacturing (CAD/CAM), using two dissimilar sacrificial instruments to 3D print a mold with the negative imprint of the scaffold design. This method directly uses additive manufacturing technology to create a biphasic scaffold consisting of bone and ligament compartments [187]. Later, polymer solutions specific to each compartment (polyglycolic acid and polycaprolactone for bone and ligament compartments, respectively) were tossed into these molds. Consequently, the solvent evaporated before getting rid of the sacrificial material. The resultant porous scaffold had defined dimensions and shape and a definite internal pore architecture. In the process of developing the two compartments independently, they were consequently gathered by utilizing a thin PCL film, hence developing into a biphasic scaffold [187]. The researchers used fibrin to deliver BMP-7-transfected human gingival fibroblasts and human periodontal fibroblasts into the bone and periodontal ligament compartments, correspondingly. The usefulness of the cellularized biphasic scaffold was monitored by means of a murine ectopic model while a human dentin block was placed in the periodontal ligament compartment. This process showed that the presence of periodontal cells to a high degree enabled the attachment of a freshly developed ligament onto the dentine slice together with the sedimentation of cementum-like tissue 6 weeks before implantation [187].
Focusing on guided bone regeneration, which is the focal point of this project, Zhang et al. (2019) have recently studied the most commonly utilized GBR membrane, known as Bio-Gide, which is among the most commonly used commercial biodegradable membranes, and has a wide range of advantages [181]. Bio-Gide possesses a bilayer makeup in which one of the sides is structured to be compact and soft to inhibit epithelium and connective tissue interference on the other side of bone defects, and the opposite side is permeable and coarse to enable the bond of osteoblasts next to the bone defect.
The aforementioned experts described a unique form of multifunctional GBR membrane with similar design characteristics as those of the Bio-Gide membrane but including extra roles that the Bio-Gide membrane cannot accomplish. The unique GBR membrane is made up of a compact nacre-like coating and a permeable membrane. The function of the nacre-like layer is to give great mechanical properties and also to inhibit non-osteoblast interference. Conversely, the porous layer has been designed with the aim of necessitating osteoblast adhesion. For a number of reasons, they asserted that their multifunctional nanocomposite membrane was better than the other GBR membranes. These reasons include biocompatibility combination with the facial surface, high mechanical performance, sufficient rate of degradation, and efficacious bacteriostasis. For these reasons, this type of nanocomposite membrane qualifies to be considered as a perfect bioactive GBR membrane for medical use [181,188].
By combining the electrospinning technique with emulsion templating, a bilayer barrier membrane (BM) made of a biodegradable synthetic polymer, PCL, was effectively developed by [189]. Some of the qualities exhibited by the resultant BM included the absence of delamination, a qualitatively resistant structure to twisting and elongation, and simplicity in handling. The electrospun layer of the BM has been proven to possess the ability to act as a barrier, offering protection to the bone defect against soft tissue interference. On the other hand, the interconnected PCL polyHIPE layer has exhibited pivotal characteristics to be the bone-enhancing layer, supplying crucial needs including boosting collagen and mineral deposition and enhancing cellular infiltration and cell compatibility [189].

Scaffold Manufacturing Technologies
Scaffolding manufacturing is a highly nuanced and evolving field, encompassing a diverse array of techniques tailored to various applications and material requirements. The process of creating porous scaffolds is far from uniform, with methodologies varying significantly in complexity, cost, and final product quality. This section delves into a selection of prevalent manufacturing technologies, each catering to specific needs and producing results unique in structure and functionality. Distinct from existing studies, such as the one referenced in citation [190][191][192][193][194][195][196][197][198][199][200], this review aims to not only present an overview of established techniques but also emphasize recent advancements, novel applications, and critical evaluations. From the rapid yet modest-quality processes to intricate and time-intensive methods that yield superior structures, we provide an insightful and contemporary analysis, with a particular focus on how these methods contribute to various applications, including bone regeneration. This comprehensive examination serves as a valuable resource for researchers and practitioners seeking an up-to-date understanding of scaffolding manufacturing technologies.

Solid Free-Form Fabrication Technique
SFFT is a manufacturing technique also recognized as rapid prototyping (RP), which also refers to a type of fabrication process called additive manufacturing [191]. In which components are printed by depositing one cross-section layer over the other layer and assembled using a three-dimensional computer-aided design (CAD) model [190]. Threedimensional scaffolds with complex geometries and dimensionally accurate structures can be manufactured using data obtained from medical scans and then adjusted to meet the needs of each individual patient [190,191].
This process is accomplished through several phases. The first phase is based on creating a computer-aided design (CAD) model, which is then sent to a file that can be manipulated with a stereolithography apparatus. Automatically, the STL file is divided into horizontal layers throughout the pre-production phase. Then, printing continues in this layered process. The final structure needs to be hardened and its surface treated before being used [191]. Through the use of sophisticated scanning techniques such as magnetic resonance imaging (MRI) or computer tomography (CT) [191]. These highly detailed 3D images can then be used to make the creation of precise [26], integrated scaffolds [192] and significantly reproducible [191]. This is particularly useful when making highly porous structures at approximately 90% or more of the total volume of the scaffold [192].
Scaffolds with sophisticated and controlled macro-and microporous structures can be provided by SFFT, potentially both within the same structure [26]. Table 4 compares the various SFFT types, which have been evaluated by different research groups [191][192][193][194]. This list includes their inherent advantages and disadvantages. SFFT is a modern development. It helps in creating solutions rather quickly, but not all types of SFFT can be used for scaffold manufacturing [191][192][193][194].  [191][192][193][194]  Complex structure, possibility of controlling porosity and size of pores independently, wide range of powder materials, solvent-free, any secondary binder system High processing temperature, using only thermally stable polymers, limited to small pore size 3D printing (3D-P) Ceramics, polymers, metals Easy process, high porosity, complete pore interconnectivity, possibility of controlling porosity and size of pores independently, macro shape control, wide range of materials Use of toxic organic solvent, lack of mechanical strength, limited to small pore size

Three-Dimensional Bioprinting Technique
Three-dimensional (3D) bioprinting is a sophisticated and intricate method of additive manufacturing that meticulously incorporates a range of biological materials to generate structures which resemble and function such as living tissues. This technique is known for its scalability, meaning it can be adjusted to create complex structures that meet individual patients' specific needs in terms of size and complexity. Furthermore, the technology enables the precise distribution of cellular components-including but not limited to growth factors, proteins, cells, and drug particles. These favorable conditions have spearheaded advancements in several medically and clinically relevant applications such as drug testing, high-throughput assays, tissue engineering, tissue regeneration, and cancer research [195].
One of the most challenging tasks in 3D bioprinting is the creation of blood vessels and organs. The complexity of this task arises from the need to integrate diverse cell types, the constraint of limited structural support, and the requirements of a concomitant capillary network, which is a typical characteristic of functional organs. Despite the considerable technological advances, these multifaceted requirements make the printing of such structures a significant challenge [196].
Nonetheless, relentless research efforts have led to some notable advancements. Researchers have successfully printed rudimentary structures, such as blood vessels, skin, and cartilage that does not require a blood supply. These achievements mark promising milestones in the field of 3D bioprinting [196]. Attempts have also been made to print bone tissue, specifically bone that comprises its natural constituents such as nerve and muscle tissue. Despite these endeavors, the structures produced are yet to match the functional superiority of their naturally occurring counterparts. Therefore, it is evident that continued research and development are needed to overcome the existing challenges in 3D bioprinting [197].

Gas-Foaming Technique
With this technique, there is no longer a need to use solvents that are normally present in the previously mentioned methods. This method creates a porous network through the dispersion of gas bubbles that, when the material is hardened, act as pores, as illustrated in Figure 5. A heated mold is used to heat the polymer material, which is usually made of polylactic-co-glycolic acid, which is then molded by compressing it to make rigid discs. After this, these molded structures are pumped with high pressure (5.5 MPa) CO 2 for 3 days at 25 • C. Afterward, gas pressure is reduced to atmospheric levels and, therefore, gas solubility is reduced. This process makes CO 2 gas create inner clumps, which then create the pores needed for the proper function of the implant. This method allows for the total number of porosities to reach up to 93% and sizes of approximately 100 mm. It is not trivial, however, to control pore size and interconnectivity with this technique [198,199].
One of the most challenging tasks in 3D bioprinting is the creation of blood vessels and organs. The complexity of this task arises from the need to integrate diverse cell types, the constraint of limited structural support, and the requirements of a concomitant capillary network, which is a typical characteristic of functional organs. Despite the considerable technological advances, these multifaceted requirements make the printing of such structures a significant challenge [196].
Nonetheless, relentless research efforts have led to some notable advancements. Researchers have successfully printed rudimentary structures, such as blood vessels, skin, and cartilage that does not require a blood supply. These achievements mark promising milestones in the field of 3D bioprinting [196]. Attempts have also been made to print bone tissue, specifically bone that comprises its natural constituents such as nerve and muscle tissue. Despite these endeavors, the structures produced are yet to match the functional superiority of their naturally occurring counterparts. Therefore, it is evident that continued research and development are needed to overcome the existing challenges in 3D bioprinting [197].

Gas-Foaming Technique
With this technique, there is no longer a need to use solvents that are normally present in the previously mentioned methods. This method creates a porous network through the dispersion of gas bubbles that, when the material is hardened, act as pores, as illustrated in Figure 5. A heated mold is used to heat the polymer material, which is usually made of polylactic-co-glycolic acid, which is then molded by compressing it to make rigid discs. After this, these molded structures are pumped with high pressure (5.5 MPa) CO2 for 3 days at 25 °C. Afterward, gas pressure is reduced to atmospheric levels and, therefore, gas solubility is reduced. This process makes CO2 gas create inner clumps, which then create the pores needed for the proper function of the implant. This method allows for the total number of porosities to reach up to 93% and sizes of approximately 100 mm. It is not trivial, however, to control pore size and interconnectivity with this technique [198,199].

Thermally Induced Phase Separation Technique
A procedure that allows for the fabrication of highly porous anisotropic scaffolds is called Thermally Induced Phase Separation (TIPS). These polymer scaffolds can be controlled with ease but have a low ability to be applied to affected tissues such as ligaments, muscles, nerves, intestines, and osseous structures [201]. Depending on the concentration of polymer used, certain characteristics will change, such as mechanical properties, pore shape, biological activity, and the rate of resorption. Furthermore, these properties will change depending on the volume of the phase separation [190]. A polymer phase fraction can be achieved by dissolving a polymer at a high degree of temperature in a certain solvent, then cooling the homogenous polymer/solvent solution to obtain a polymer porous scaffold. After this process is completed, a microporous scaffold can be obtained immediately after the solvent has evaporated, as schematically shown in Figure 6 [201].
A procedure that allows for the fabrication of highly porous anisotropic scaffolds is called Thermally Induced Phase Separation (TIPS). These polymer scaffolds can be controlled with ease but have a low ability to be applied to affected tissues such as ligaments, muscles, nerves, intestines, and osseous structures [201]. Depending on the concentration of polymer used, certain characteristics will change, such as mechanical properties, pore shape, biological activity, and the rate of resorption. Furthermore, these properties will change depending on the volume of the phase separation [190]. A polymer phase fraction can be achieved by dissolving a polymer at a high degree of temperature in a certain solvent, then cooling the homogenous polymer/solvent solution to obtain a polymer porous scaffold. After this process is completed, a microporous scaffold can be obtained immediately after the solvent has evaporated, as schematically shown in Figure 6 [201].

Emulsion Freeze-Drying Technique
This technique is based on the phase fraction through the use of different physical properties of the fiber by emulsifying the solution and then drying it at a very low temperature [202], and producing a scaffold that has abundant pores, as illustrated in Figure  7.

Emulsion Freeze-Drying Technique
This technique is based on the phase fraction through the use of different physical properties of the fiber by emulsifying the solution and then drying it at a very low temperature [202], and producing a scaffold that has abundant pores, as illustrated in Figure 7.
ately after the solvent has evaporated, as schematically shown in Figure   Figure 6. Schematic representation of the porous scaffold fabrication process w Induced Phase Separation (TIPS), [source: [200], redesigned with copyright per vier license number: 4724150945567, dated 8 December 2019].

Emulsion Freeze-Drying Technique
This technique is based on the phase fraction through the use of d properties of the fiber by emulsifying the solution and then drying it a perature [202], and producing a scaffold that has abundant pores, as illu 7. The first step in this process is the creation of the emulsion by homogenizing a polymer in a carbon-based solvent and water. This emulsion must be quickly frozen and the formed phases (solvent and water) are then eliminated by freeze-drying the sample. The resulting polymer scaffolds will have pores of between 20 and 200 µm [190]. This method could be combined with the third method that was mentioned, as well as adding crystal-forming polar compounds such as sucrose or NaCl, in order to further increase porosity. Once the sample has been dried, these particles can be cleared with the use of water [167].

Solvent Casting and Particulate Leaching Technique
Another common method for making scaffolds is solvent casing. This process starts with the deconstruction of a polymer in a carbon-based solvent, as schematically shown in Figure 8. The aforementioned method uses "porogens", a group of chemical compounds that can be distributed into a structure during the manufacturing process and then taken away through the use of water, leaving behind a porous structure. These porogens can create a coupled polymer-porogen structure when added to the overall solution. As soon as the polymer reaches its final form and starts hardening, and the original solvent evaporates away, water is then used to dissolve porogens, which is often a high polarity compound, such as NaCl. Although it is hard to control the final inner structure of the scaffold since it is difficult to predict and control where the porogen particles will be distributed and then dissolved, a three-dimensional porous polymer scaffold was obtained [199,203].
The first step in this process is the creation of the emulsion by homogenizing a p mer in a carbon-based solvent and water. This emulsion must be quickly frozen and formed phases (solvent and water) are then eliminated by freeze-drying the sample resulting polymer scaffolds will have pores of between 20 and 200 µm [190]. This me could be combined with the third method that was mentioned, as well as adding cry forming polar compounds such as sucrose or NaCl, in order to further increase poro Once the sample has been dried, these particles can be cleared with the use of water [

Solvent Casting and Particulate Leaching Technique
Another common method for making scaffolds is solvent casing. This process s with the deconstruction of a polymer in a carbon-based solvent, as schematically sh in Figure 8. The aforementioned method uses "porogens", a group of chemical pounds that can be distributed into a structure during the manufacturing process and taken away through the use of water, leaving behind a porous structure. These poro can create a coupled polymer-porogen structure when added to the overall solution soon as the polymer reaches its final form and starts hardening, and the original so evaporates away, water is then used to dissolve porogens, which is often a high pol compound, such as NaCl. Although it is hard to control the final inner structure o scaffold since it is difficult to predict and control where the porogen particles will be tributed and then dissolved, a three-dimensional porous polymer scaffold was obta [199,203].

Spin-Coating Technique
The spin coating technique is a widely used method for depositing thin films of terials onto a flat substrate. In this technique, a liquid solution containing the scaffold terial is dispensed onto the substrate, which is then rotated at high speeds (typically i range of 1000 to 4000 rpm). The centrifugal force generated by the spinning subs causes the solution to spread out evenly over the substrate, forming a thin film. The s and duration of the spinning process can be controlled to achieve a desired thickness uniformity of the film.
In Figure 9, a schematic outline of the spin coating process is shown. The figur picts the substrate, the spin coater, and the solution being applied to the substrate. A

Spin-Coating Technique
The spin coating technique is a widely used method for depositing thin films of materials onto a flat substrate. In this technique, a liquid solution containing the scaffold material is dispensed onto the substrate, which is then rotated at high speeds (typically in the range of 1000 to 4000 rpm). The centrifugal force generated by the spinning substrate causes the solution to spread out evenly over the substrate, forming a thin film. The speed and duration of the spinning process can be controlled to achieve a desired thickness and uniformity of the film.
In Figure 9, a schematic outline of the spin coating process is shown. The figure depicts the substrate, the spin coater, and the solution being applied to the substrate. As the substrate is spun, the centrifugal force pulls the solution towards the edges, creating a thin and uniform film on the substrate.
Polymers 2023, 15, x FOR PEER REVIEW 25 substrate is spun, the centrifugal force pulls the solution towards the edges, creating a and uniform film on the substrate. The advantages of the spin coating technique include its simplicity and cost-e tiveness, as well as its ability to produce films with precise control over their thick and uniformity. This technique can also be easily combined with other techniques to ate complex multilayered structures, such as for periodontal tissue regeneration.

Limitations of the preceding manufacturing techniques
In practice, the techniques used to manufacture scaffolds are divided into solid form fabrication and conventional methods. Each of them produces various scaffolds distinctive characteristics [204]. Even though SFFT provides a plethora of potential op tunities for tissue engineering and possesses undeniable advantages, there are som herent drawbacks that must be considered. Firstly, each method uses a very specific rication material. SLS uses a fine powder, whereas the use of thermoplastics is more cient for FDM. Even when the selected material is appropriate, if it is difficult to pre it can make the whole process much more challenging. Secondly, the fact that a mat can be successfully printed does not guarantee its proper function, since successful folds also require constructs to maintain their integrity throughout their layers. The terial must be able to support itself after its fabrication, maintaining its integrity laye layer. Thirdly, in the case of the printing of biological tissues, novel material solidifica techniques that are used to preserve the fabricated scaffold integrity should be devel [205]. Lastly, when using materials that are cell-loaded, the flexibility of print param such as shear stress or temperature is restricted. This happens since cell environment ever changing, and doing so would be deleterious for cell survival [206].
While conventional techniques of scaffolding fabrication include the constructio porous polymer structures such as substrates for cell adhesion, it is difficult to obtain plex structures with tunable microscale and macroscale using conventional methods [ In addition, some of these methods are manual based. Therefore, they are labor inten and difficult to reproduce. Another limitation is the need for organic solvents and p gens, which are cytotoxic and their residues may cause inflammatory responses [ Benefits and limitations of conventional and Solid free-form manufacturing techni are discussed and summarized in Table 5 [188,209]. The advantages of the spin coating technique include its simplicity and cost-effectiveness, as well as its ability to produce films with precise control over their thickness and uniformity. This technique can also be easily combined with other techniques to create complex multilayered structures, such as for periodontal tissue regeneration.

Limitations of the preceding manufacturing techniques
In practice, the techniques used to manufacture scaffolds are divided into solid freeform fabrication and conventional methods. Each of them produces various scaffolds with distinctive characteristics [204]. Even though SFFT provides a plethora of potential opportunities for tissue engineering and possesses undeniable advantages, there are some inherent drawbacks that must be considered. Firstly, each method uses a very specific fabrication material. SLS uses a fine powder, whereas the use of thermoplastics is more efficient for FDM. Even when the selected material is appropriate, if it is difficult to prepare, it can make the whole process much more challenging. Secondly, the fact that a material can be successfully printed does not guarantee its proper function, since successful scaffolds also require constructs to maintain their integrity throughout their layers. The material must be able to support itself after its fabrication, maintaining its integrity layer by layer. Thirdly, in the case of the printing of biological tissues, novel material solidification techniques that are used to preserve the fabricated scaffold integrity should be developed [205]. Lastly, when using materials that are cell-loaded, the flexibility of print parameters such as shear stress or temperature is restricted. This happens since cell environments are ever changing, and doing so would be deleterious for cell survival [206].
While conventional techniques of scaffolding fabrication include the construction of porous polymer structures such as substrates for cell adhesion, it is difficult to obtain complex structures with tunable microscale and macroscale using conventional methods [207]. In addition, some of these methods are manual based. Therefore, they are labor intensive and difficult to reproduce. Another limitation is the need for organic solvents and porogens, which are cytotoxic and their residues may cause inflammatory responses [208]. Benefits and limitations of conventional and Solid free-form manufacturing techniques are discussed and summarized in Table 5 [188,209]. Over the last ten years, electrospinning technology has become one of the most interesting methods for creating scaffolds used for tissue engineering. The creation of these nanofiber scaffolds has become the focus of research for many investigators due to their many unique properties, especially those which are clinically relevant. In particular, this method is used in order to manufacture nanofibers used in different applications in dentistry such as tooth restoration, repair of oral mouth tissue, preventing tooth decay, and restoring other dental and periodontal tissues, such as the repair of dentin, endodontium, oral mucosa, periodontal tissue, as well as alveolar bone regeneration [210].
A type of material that has received a lot of attention recently has been biodegradable polymers, especially in biomedical areas such as bio-prosthetics, tissue engineering, and the application of drug delivery systems. Aliphatic polyesters are one of the most significant types of synthetic biodegradable polymers, owing in particular to their advantageous characteristics of biocompatibility and biodegradability. The main allure of these polymers (polyesters) is their biological compatibility and their ability to be degraded within the organ [211].
Electrostatic spinning, most commonly known as electrospinning, has been a focal point of research for the last 20 years due to the various potential uses of the created microfiber in both nanotechnologies and nanoscience [212]. Notable characteristics, such as high permeability, large surface-to-volume ratio, and excellent pore interconnection, have made electrospun microfiber ideal for normal cell functions, such as nutrient and cell transportation [213,214].
Additionally, the nanofibrous scaffolds manufactured through this method can provide excellent extracellular conditions, such as coupling, migration, and cell proliferation, especially for those cells in charge of hard tissue repair. Along with the simplicity of setup and cost efficiency, the opportunity to create microfiber with a large variety of physical and chemical properties is its own merit. This machine consists of a syringe needle, a grounded collector (metal plate), a high-voltage electrical source, and a syringe pump ( Figure 10). many unique properties, especially those which are clinically relevant. In particular method is used in order to manufacture nanofibers used in different applications in tistry such as tooth restoration, repair of oral mouth tissue, preventing tooth decay, restoring other dental and periodontal tissues, such as the repair of dentin, endodont oral mucosa, periodontal tissue, as well as alveolar bone regeneration [210].
A type of material that has received a lot of attention recently has been biodegrad polymers, especially in biomedical areas such as bio-prosthetics, tissue engineering the application of drug delivery systems. Aliphatic polyesters are one of the most sig cant types of synthetic biodegradable polymers, owing in particular to their advantag characteristics of biocompatibility and biodegradability. The main allure of these p mers (polyesters) is their biological compatibility and their ability to be degraded w the organ [211].
Electrostatic spinning, most commonly known as electrospinning, has been a point of research for the last 20 years due to the various potential uses of the created crofiber in both nanotechnologies and nanoscience [212]. Notable characteristics, su high permeability, large surface-to-volume ratio, and excellent pore interconnection, made electrospun microfiber ideal for normal cell functions, such as nutrient and transportation [213,214].
Additionally, the nanofibrous scaffolds manufactured through this method can vide excellent extracellular conditions, such as coupling, migration, and cell prolifera especially for those cells in charge of hard tissue repair. Along with the simplicity of s and cost efficiency, the opportunity to create microfiber with a large variety of phy and chemical properties is its own merit. This machine consists of a syringe need grounded collector (metal plate), a high-voltage electrical source, and a syringe p ( Figure 10). The electrical source must carry around 10 to 30 kilovolts and is applied to solu that are ejected via the syringe needle. When the electrical charge reaches the sta point, the surface tension of the charged solution begins to change, causing a deform The electrical source must carry around 10 to 30 kilovolts and is applied to solutions that are ejected via the syringe needle. When the electrical charge reaches the starting point, the surface tension of the charged solution begins to change, causing a deformation of the solution droplet into a conical droplet known as the Taylor cone. While the electrical force overcomes the surface tension of the charged solution, thin charged jets are ejected from the tip of the metallic needle in a nearly straight line towards the electrically inverse electrode. As the material is being extruded, the solvent is being evaporated away, resulting in the construction of continuous dry polymer fiber, which leads to the formation of a non-woven surface of the obtained fiber. The grounded collector surface is generally placed approximately 20 cm from the syringe's tip [215].

Future Directions and Conclusions
The dynamic landscape of guided tissue and bone regeneration is primarily driven by the interplay of biomaterials and scaffold manufacturing technologies, offering a diverse array of solutions for periodontal therapeutic interventions. As we move forward, several trends and directions can be envisioned that are expected to shape the future trajectory of this field.
One such direction is the exploration of more advanced, next-generation membranes, which could potentially surpass the benefits offered by the current third-generation membranes. The development of these membranes could focus on enhanced biocompatibility, optimized resorption rates, and improved mechanical strength. For instance, research on Floreon™ blend and other innovative polymer combinations that offer superior mechanical strength and enhanced bioresorbability may provide novel solutions for effective tissue regeneration [124]. A focus on personalized treatment approaches utilizing patient-specific 3D bioprinting technologies may also prove beneficial [195].
Furthermore, the integration of growth factors or other bioactive molecules within these membranes to promote bone and tissue regeneration is a promising area of exploration. With recent advances in drug delivery technologies, the controlled and localized delivery of these factors could significantly enhance therapeutic outcomes [49,170]. A notable recent development is the use of Zn-based biodegradable materials in biomedical applications. With their potential to serve as next-generation orthopedic implants, Zn-based materials may offer significant advantages over conventional alternatives, such as reducing the need for revision surgeries and minimizing biocompatibility issues [216]. These materials have demonstrated a significant role in bone metabolism and new cell growth, and they show medium degradation without the release of excessive hydrogen. The addition of alloying elements such as Mg, Zr, Mn, Ca, and Li into pure Zn has been found to enhance the mechanical properties of Zn alloys, making them a promising material for future guided tissue and bone regeneration applications [216]. Additionally, there is an increasing interest in nanotechnologies and their application in bone regeneration. The fabrication of nanocomposite scaffolds can potentially improve the cellular response and lead to better mimicking of the natural extracellular matrix. Such improvements could foster bone formation and promote better integration with host tissue [50,181].
In the realm of scaffold manufacturing technologies, there is considerable scope for enhancing precision and reducing production times, perhaps through the advancement of computer-aided design and manufacturing (CAD/CAM) techniques. Hybrid fabrication methods that combine the advantages of different techniques can also be explored, such as combining electrospinning with 3D printing, to create complex scaffold architectures with improved mechanical properties and porosity [187].
The future direction of guided tissue and bone regeneration is also increasingly being influenced by the emerging field of tissue engineering, specifically the use of stem cells. Combining biomaterials with stem cell therapy could lead to unprecedented advancements in periodontal tissue regeneration, offering high-potential treatment strategies that need to be explored extensively [217].
In conclusion, guided tissue and bone regeneration represents an exciting and rapidly advancing field with vast potential for further innovation and improvement. The progression in this field will rely heavily on a comprehensive understanding of biomaterials and their interactions with biological systems, as well as the continued refinement of manufacturing technologies. Embracing an interdisciplinary approach, encompassing materials science, biology, engineering, and clinical dentistry will be essential for the successful evolution of periodontal treatments.