Healthy bone has the unique ability to spontaneously regenerate. However, if the diseased or damaged area exceeds a certain size, bone grafting is needed to regenerate the tissue [1
]. Common practices to supplement bone regeneration in larger defects include bone graft biomaterials such as autograft (patient bone), allograft (human cadaver bone), xenograft (animal bone), and synthetic biomaterials (i.e.
, ceramics, cements, glasses, metals, polymers, and composites) [2
]. Since each of the above mentioned grafts have their own distinct disadvantages, synthetic biomaterial scaffolds that are biocompatible, biodegradable, porous, bioactive, and mechanically stable have been the focus of research as alternative bone substitutes.
Collagen and hydroxyapatite (HA) are popular materials when investigating bone scaffolds since their composites mimic the extracellular matrix (ECM) of natural bone [7
]. More recently, gelatin (denatured collagen) has been used as a replacement for collagen since it is less expensive, easier to obtain, and contains similar functional groups which enhance cellular response [12
]. Freeze-dried gelatin sponges have many advantages because they can be designed to fit any size defect/injury site, have the ability to swell and fill a void space, degrade controllably in a range of rates (due to various cross-linking methods) to ensure drug release and mechanical stability, and can be easily modified by incorporating various osteoconductive/osteoinductive materials (i.e.
, minerals, growth factors, proteins, etc.
). The addition of bioactive inorganic HA to freeze-dried gelatin sponges creates a bone-like ECM scaffold which allows a more controlled drug delivery system and increases cellular attachment, proliferation, alkaline phosphatase activity, and osteocalcin production [13
]. HA also has the ability to bind to a variety of molecules, including proteins. As a result, scaffolds incorporated with HA provide a more favorable environment through increased adsorption of serum adhesion protein such as fibronectin and vitronectin [16
]. Enhanced cellular responses have also been observed with the addition of other minerals (β-tricalcium phosphate, Dicalcium Phosphate Dihydrate), polymers (gellan, poly-lactide-co-glycolide) and proteins (bone morphogenetic protein, Wnt1 inducible signaling pathway protein) in combination with HA [17
Other additions such as chitin whiskers (CW, i.e.
, chitin nanocrystals) and platelet-rich plasma (PRP) have been used to increase the mechanical integrity, bioactivity, and osteogenic potential of scaffolds. In recent years, experiments studying CW have increased due to its availability, nontoxicity, and ability to mechanically reinforce polymer nanocomposites and enhance cell proliferation [20
]. Results of numerous studies have demonstrated the versatility and effectiveness of PRP within wound healing, skin engineering, ligament/tendon engineering, cartilage repair, bone regeneration, and more [26
]. Particularly with bone regeneration, the addition of PRP has been found to increase bone density/mineralization, vascularization, and osteogenesis [30
]. Preparation rich in growth factors (PRGF, a bioactive lyophilized version of PRP) contains high concentrations of growth factors such as platelet-derived growth factor (PDGF), transforming growth factor-β (TGF-β), insulin-like growth factor-1 (IGF-1), vascular endothelial growth factor (VEGF), epidermal growth factor (EGF), fibroblast growth factor (FGF), and more [37
]. PRGF also contains cell adhesive proteins such as fibronectin and vitronectin. In relation to bone remodeling, these growth factors and proteins elicit a favorable cellular response which supports the incorporation of PRGF within scaffolds intended for bone tissue engineering [40
]. The bone remodeling functions for some of these molecules are summarized in Table 1
. The incorporation of PRGF within gelatin sponges, the general combination of CW and PRGF, and the combination of PRGF with HA and/or CW in gelatin sponges are all areas that have yet to be explored.
Role of PRGF components in bone remodeling [37
Role of PRGF components in bone remodeling [37,38,39,40,41,42,43].
|PDGF||Mesenchymal stem cell and progenitor cell recruitment, proliferation, migration and differentiation into osteoblasts|
|TGF-β||Mesenchymal stem cells differentiation, increased production of collagen and mineral matrix|
|IGF-1||Osteoblast proliferation and differentiation|
|VEGF||Angiogenesis, Endochondral ossification|
|Fibronectin, Vitronectin||Enhance formation of focal adhesions by osteoblasts, osteoblast migration|
The present study aimed to evaluate the release kinetics, degradation, mechanical properties, and cellular responses of multiple combinations of composite freeze-dried gelatin sponges. PRGF, CW, and/or HA were incorporated into the gelatin sponges and cross-linked during gelation or after lyophilization to increase the scaffolds’ overall compatibility as a bone tissue engineering substitute.
3. Experimental Section
3.1. Fabrication of Gelatin Composite Sponges
All scaffolds were fabricated with a base solution of 30 mg/mL gelatin (Type B from Bovine skin, Sigma) in deionized (DI) water. For composite scaffolds, a total amount of 10 mg/mL of PRGF, HA, and/or CW were weighed, added to the 30 mg/mL gelatin solution, then sonicated if necessary (Table 5
). Materials included HA nanopowder (particle size <
200 nm (BET), Sigma-Aldrich), CW (prepared by following a published protocol [22
]), PRGF (created using published protocol [38
]), and1-Ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride (EDC, Thermo Scientific). To create PRGF, fresh human whole blood from 3 donors was purchased, combined, and centrifuged (SmartPReP®
2) to create PRP. PRP then underwent a freeze (−70 °C)–thaw (37 °C)–freeze (−70 °C) cycle to ensure platelet lysis and activation. Frozen PRP was then lyophilized to obtain a dry PRGF powder to be weighed and incorporated within the gelatin solution [38
Scaffold Components and Fabrication Concentrations.
Scaffold Components and Fabrication Concentrations.
|Amount (mg/mL) Added to Gelatin Solution||Sonicated|
|+HA+CW||5 (HA) and 5 (CW)||yes|
|+PRGF+HA||5 (PRGF) and 5 (HA)||yes (HA) then PRGF added|
|+PRGF+CW||5 (PRGF) and 5 (CW)||yes (CW) then PRGF added|
|+PRGF+HA+CW||3.33 (PRGF), 3.33 (HA), and 3.33 (CW)||yes (HA+CW) then PRGF added|
4 mL of the prepared gelatin or gelatin composite solution was pipetted into a 35 × 10 mm Petri dish, refrigerated at 4 °C overnight to gel, and then slowly frozen at −15 °C overnight, −20 °C for 4 h, and −70 °C for 4 h. Frozen gel composites were lyophilized for 24 h then cross-linked for 18 h at room temperature in 50 mM EDC in ethanol [45
]. To compare cross-linking methods, another set of solutions were made and 50 mM EDC was added directly to the composite solution before refrigerating to gel, the following steps remained the same as previously described (scaffolds denoted as +EDC). Using a Miltex biopsy punch, 6 mm discs were punched and used for all experiments.
3.2. Mass Loss
Two 6 mm scaffold punches were weighed as a unit for initial dry mass. Scaffolds were then disinfected (30 min ethanol followed by three 10 min washes of PBS) and transferred to a 48 well plate (two discs per well, n = 3). 500 µL of Dulbecco’s modified Eagle’s medium (DMEM) high glucose containing 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin was added to each well. The two scaffolds per well were incubated in media at 37 °C and 5% CO2 with media changes every 7 days. Scaffolds were removed and weighed as a unit every 7 days up to 90 days. Hydrated scaffolds were massed and compared to original dry weights as a percentage to determine percent increase in scaffold mass. Scaffolds were air-dried post 90 day culture and compared to original dry weights to determine overall mass loss.
3.3. Protein Release
To determine total protein content of each 6 mm scaffold disc, triplicates of one non cross-linked 6 mm discs of each scaffold type was immersed in 500 µL of 1× PBS. Uncross-linked scaffolds completely degraded within minutes at room temperature. Since scaffolds are primarily comprised of gelatin, the degraded byproducts are detectable using a general protein assay. Protein was quantified using a Pierce BCA Protein Assay (Thermo Scientific). Briefly, 25 µL of PBS containing the degraded scaffold contents was added to 200 µL of working reagent in a 96-well plate. The well plate was then incubated at 37 °C for 30 minutes, cooled to room temperature, and absorbance measured at 562 nm using a SpectraMax Plus 384 Microplate Spectrophotometer (Molecular Devices).
Scaffold release kinetics was studied by quantifying protein release from each scaffold over a period of 90 days. Triplicates of one 6 mm disc of each cross-linked scaffold type were incubated in 500 µL of 1× PBS at 37 °C with PBS replaced every 3 days. After 1, 4, 7, 10, 14, 21, 28, 56, and 90 days the PBS containing released scaffold contents in each well was analyzed for protein content using the Pierce BCA Protein Assay described above.
3.4. Cell Attachment, Migration and Matrix Production
Triplicates of 6 mm discs of each scaffold composition were seeded with 50,000 osteoblast-like cells (MG-63 cells from a human osteosarcoma) and incubated at 37 °C and 5% CO2 in DMEM high glucose media containing 10% FBS and 1% penicillin/streptomycin with media changes every three days. After 1, 28, and 90 days, scaffolds were fixed in 10% formalin and stored at 4 °C until preparation for scanning electron microscopy (SEM) and fluorescent staining. For uniaxial compression testing, scaffolds were tested directly after being removed from the incubator.
3.4.1. Scanning Electron Microscopy
Scaffolds were removed from formalin, briefly rinsed in PBS and water, and then subjected to ethanol dehydration (10 min soaks in 30, 50, 70, 90, and 100% ethanol, subsequently). Samples were air dried overnight, mounted on aluminum stubs, sputter coated in gold for 70 sec, and examined using a JEOL JSM-5610LV scanning electron microscope.
3.4.2. DAPI Staining
Scaffolds were removed from formalin, immersed in a 30% sucrose solution in DI water for 48 hours at 4 °C to ensure displacement of all air bubbles, suspended in premium frozen section compound (VWR), and frozen at −70 °C overnight. 60 µm slices were cryosectioned using a Cryostat (Thermo) and transferred to microscope slides. Cryosectioned samples were then stained with 4'-6-diamidino-2-phenylindole (DAPI) stain for 5 min and imaged using a UV fluorescent microscope to display the location of cell nuclei.
3.4.3. Alizarin Red S Staining
Alizarin Red S (ARS) is a dye that selectively binds to calcium salts. ARS staining was used to quantify scaffold mineral content by modifying a published protocol [49
]. ARS was performed on the 6 mm scaffold punches after 1, 28, and 90 days incubation in media with and without cells. After incubation scaffolds were stained with 40 mM alizarin red for 30 min then washed with DI water to remove any unbound stain. Scaffolds were then transferred to a 2 mL microcentrifuge tube containing 1.5 mL of 50% acetic acid to destain for 1 h at room temperature. 500 µL of the solubilized stain was added to 600 µL of 1 M NaOH to adjust the pH to 4.1. 200 µL of this solution was pipetted into a 96-well plate and absorbance read at 550 nm using a SpectraMax Plus 384 Microplate Spectrophotometer (Molecular Devices).
3.5. Uniaxial Compression Testing
Uniaxial compression testing was performed on acellular and cellularized 6 mm scaffold discs after 1, 28, and 90 days incubation in media. Mechanical testing was conducted by attaching an indenter (cylindrical, 2 mm diameter, plane-ended, stainless steel) to a MTS Bionix 200 Mechanical Testing System instrument with a 100 N load cell (MTS Systems Corp., USA). Indentation was performed perpendicular to the scaffold surface at the center of each scaffold disc. The discs were placed on a flat metal surface and kept hydrated with PBS. The indenter was lowered to the surface of the scaffolds and the following parameters were used: test speed of 0.5 mm/min, data acquisition rate of 10 Hz, a preload of 0.015 N, and a max indenter displacement of 90% of the scaffold thickness. Peak load was calculated by the MTS software TestWorks 4.0. Many scaffolds at later time points did not register a preload until later in the testing which resulted in the indenter moving through the entire scaffold until reaching the maximum load of 100 N when the intender contacted the metal plate. In these instances, the maximum peak load plateau, just before the maximum load was reached, was extracted from the graph and reported.
3.6. Statistical Analysis
Statistical analysis was performed using JMP IN 9 statistical software (SAS Institute) to determine significant differences. Analysis of the data was based on a Kruskal-Wallis one-way analysis of variance on ranks and a Tukey-Kramer pairwise multiple comparison procedure. The results are presented in mean ± standard deviation (SD). Unless otherwise specified all samples were run at a minimum of triplicate (n = 3) to ensure statistical significance.
In this study it was demonstrated that a lyophilized gelatin gel sponge, modified through the addition of PRGF, HA, and CW, demonstrated clear osteogenic potential when cultured with an MG-63 osteoblast-like cell line. These scaffolds, further modified through EDC cross-linking during gelation, were able to remain intact after 90 days in culture while exhibiting a controlled protein release. This tailorable rate of degradation is critical in a bone repair scaffold, where scaffold breakdown needs to match the ingrowth of new bony matrix to prevent catastrophic failure or the potential for micromotion or stress-shielding to occur. While this preliminary study failed to determine a clear optimal combination of gelatin and scaffold modifying agents (PRGF, HA, CW) to promote bone regeneration, it demonstrated that the use of a lyophilized gelatin gel sponge with the potential to absorb several times its weight in water was capable of eliciting cell infiltration into the structures as well as promoting the formation of cell-created mineral matrix. However, the +EDC scaffolds containing +PRGF+HA+CW performed well in all preliminary evaluations and need to be further investigated. Testing must be performed to more accurately determine the cellular interaction with these scaffolds, particularly the cellular response inside the structures.