Search Results (13)

Effective chronic skin wound healing remains challenging due to excessive inflammation, insufficient vascular support, and impaired extracellular matrix remodeling. By rationally designing and integrating complementary biomaterials, it is possible to synergistically tailor physicochemical properties and biological performance for tissue repair and regeneration. In this study, a gelatin-based composite hydrogel incorporating recombinant type III collagen (rColIII) and carboxymethyl cellulose (CMC) was developed via EDC/NHS-mediated crosslinking and evaluated for wound repair. By tuning the rColIII/CMC ratio, the hydrogel mechanical modulus (G′) increased from ~1.2 kPa to ~2.6 kPa, and enzymatic degradation could be modulated, as reflected by changes in the remaining material mass. The optimized Gel/rCol/CMC-1 formulation supported 3T3 cell migration (1.8-fold increase at 24 h) and promoted a pro-regenerative (M2-like) macrophage phenotype in vitro. In a full-thickness diabetic wound model, Gel/rCol/CMC-1 accelerated wound closure (82.3 ± 4.7% vs. 56.9 ± 5.1% at day 14) and enhanced tissue quality, evidenced by more organized collagen deposition and increased CD31⁺/α-SMA⁺ vessel density. These results demonstrate that formulation-driven tuning of gelatin/rColIII/CMC matrices creates a supportive microenvironment for coordinated wound repair, highlighting their potential as regenerative hydrogel dressings for difficult-to-heal wounds. Full article

Synthesized by expressing natural collagen sequences in specific hosts, recombinant collagen exhibits multiple advantages, encompassing a higher content of bioactive domains, enhanced antioxidant activity, the absence of viral pathogens, favorable hydrophilicity, reproducible production, and low immunogenicity. Consequently, it has found extensive use in applications ranging from biomaterials and pharmaceuticals to skincare. This review systematically explores various expression systems for recombinant collagen, including those utilizing Escherichia coli, Pichia pastoris, plants, insect baculovirus, and mammalian cells. It provides a detailed comparison of their differences and commonalities in terms of production efficiency, post-translational modification capability, and cost-effectiveness. Key separation and purification techniques for recombinant collage-notably precipitation, affinity chromatography, ion-exchange chromatography, and gel filtration chromatography are further introduced, with an in-depth analysis of the applicable scenarios and purification outcomes for each method. Finally, the review comprehensively summarizes the characterization methods for both the physicochemical properties and biological functions of recombinant collagen. For physicochemical properties, techniques covered include scanning electron microscopy, micro-differential thermal analysis, circular dichroism spectroscopy, SDS-PAGE, mass spectrometry, and Fourier-transform infrared spectroscopy. For biological functions, the focus is on its roles and the corresponding assessment methods in processes such as cell proliferation, migration, adhesion, and wound healing. Building upon this comprehensive overview, current challenges facing recombinant collagen are identified, and future directions are proposed, emphasizing the need to reduce R&D costs, refine testing methods for cosmetic products, and improve safety evaluation protocols to advance the field. Full article

Recombinant human bone morphogenetic protein-2 (rhBMP-2) is widely used to promote bone regeneration. However, conventional surface-attached delivery on absorbable collagen sponges causes a rapid burst release, excessive inflammation, and suboptimal healing. To overcome these limitations, we developed a thermally controlled Poly(DL-lactide-co-glycolide) (P_DLLGA) encapsulation system, designed to stabilize rhBMP-2 and enable controlled release. rhBMP-2 was incorporated in P_DLLGA pellets using the hot-melt extrusion of a lyophilized mixture containing poloxamer 407 and hydroxypropyl-β-cyclodextrin, and terminal sterilization (X-ray irradiation). The released rhBMP-2 maintained its molecular integrity after sterilization and remained stable for up to 732 days in storage, as confirmed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and capillary electrophoresis (CE). Further, high-affinity binding between released rhBMP-2 and BMPR-IA was confirmed by bio-layer interferometry (BLI), and the released protein induced a robust in vitro ALP response, confirming preserved osteogenic activity. Our encapsulation approach for rhBMP-2 using P_DLLGA, including the combination product with β-TCP (LDGraft; Locate Bio, Nottingham, UK), provides a stable and bioactive rhBMP-2 delivery strategy with inherent dose-shielding properties, supporting safe, controlled, and effective bone regeneration therapies. Full article

The purpose of this study is to achieve the quantitative detection of recombinant type III collagen (rh-COL-III) in dressings with complex matrix. First of all, the marker peptide (GEAGIPGVPGAK) of rhCOL-III was identified with HPLC-MS/MS. Then, a qualitative and quantitative method based on marker peptides was established and validated. In order to obtain higher sensitivity, a pretreatment method of liquid, gel, and ointment dressings was optimized. The reference material for quantification was combined using rhCOL-III and blank matrix of each dressing. The results indicated that the relative standard deviation (RSD) of the quantitative method was 2.77%, and the RSD of intraday and interday precision was 2.76% and 2.31%, respectively. The spiking recovery rate was between 80% and 90%. The optimal pretreatment method was Tris-HCl solvent replacement. The optimal trypsin concentration for the dressing solution was 20 μg in 500 μL. The method of preparing standard substances with a blank matrix can effectively eliminate the influence of the matrix effect on the quantitative results. The average spiking recovery rates of 50 μg/mL, 100 μg/mL, and 200 μg/mL in three different dressings ranged from 80% to 120%. The quantitative detection of limit (LOD) of rhCOL-III was 1 ng/mL, 2 ng/g, and 1 ng/g in liquid, ointment, and gel dressings. Full article

Excessive ultraviolet (UV) exposure can lead to sunburn, characterized by skin barrier damage, inflammation, pain, and an increased risk of skin cancer. Recombinant collagens have gradually attracted attention due to their high purity, low immunogenicity, batch-to-batch consistency, and excellent solubility. Additionally, the type of dressing significantly affects wound repair. Gels are ideal for sunburn treatment because they maintain a moist environment, adhere firmly, and do not need to be removed. Herein, we have created bioactive triple-helical recombinant collagen (THRC) gels for improved healing of sunburned skin. The THRC gels remained stable after a three-month stability test, displaying a rheological behavior characteristic of non-Newtonian pseudoplastic fluids. In vivo skin irritation tests conducted on New Zealand rabbits demonstrated that THRC gels were safe for use. A sunburned mice model was established to study the biological effects of THRC gels. Non-invasive combo evaluations indicated that THRC gels exhibited an exceptional capability of recovering dermis density, erythema index (EI), hydration, and transepidermal water loss (TEWL) of sunburned skin to a healthy state. Histological observations revealed that THRC gels significantly enhanced the repair of damaged skin by accelerating the recovery process, promoting collagen deposition and regeneration. Molecular biological characterizations further demonstrated their remarkable antioxidant properties, including the inhibition of lipoperoxidation and the enhancement in superoxide dismutase (SOD) and glutathione (GSH) activities. These safe and bioactive recombinant collagen gels provide a novel approach for sunburn repair and show potential for long-term cosmetic benefits. Full article

At present, only a few reports have addressed the possible contribution of the lymphatic vascular system to the pathogenesis of systemic sclerosis (SSc). Based on the evidence that blood vascular endothelial cells can undertake the endothelial-to-myofibroblast transition (EndMT) contributing to SSc-related skin fibrosis, we herein investigated whether the lymphatic endothelium might represent an additional source of profibrotic myofibroblasts through a lymphatic EndMT (Ly-EndMT) process. Skin sections from patients with SSc and healthy donors were immunostained for the lymphatic endothelial cell-specific marker lymphatic vessel endothelial hyaluronan receptor-1 (LYVE-1) in combination with α-smooth muscle actin (α-SMA) as the main marker of myofibroblasts. Commercial human adult dermal lymphatic microvascular endothelial cells (HdLy-MVECs) were challenged with recombinant human transforming growth factor-β1 (TGFβ1) or serum from SSc patients and healthy donors. The expression of lymphatic endothelial cell/myofibroblast markers was measured by quantitative real-time PCR, Western blotting and immunofluorescence. Collagen gel contraction assay was performed to assess myofibroblast-like cell contractile ability. Lymphatic endothelial cells in intermediate stages of the Ly-EndMT process (i.e., coexpressing LYVE-1 and α-SMA) were found exclusively in the fibrotic skin of SSc patients. The culturing of HdLy-MVECs with SSc serum or profibrotic TGFβ1 led to the acquisition of a myofibroblast-like morphofunctional phenotype, as well as the downregulation of lymphatic endothelial cell-specific markers and the parallel upregulation of myofibroblast markers. In SSc, the Ly-EndMT might represent a previously overlooked pathogenetic process bridging peripheral microlymphatic dysfunction and skin fibrosis development. Full article

Leucine-rich alpha-2-glycoprotein 1 (LRG1) mediates skin repair and fibrosis by stimulating the transforming growth factor-beta (TGF-β) signaling pathway. In the present study, we investigated the effect of LRG1 on extracellular matrix (ECM) integrity in fibroblasts, as well as on skin aging. The treatment of dermal fibroblasts with purified recombinant human LRG1 increased type I collagen secretion and decreased matrix metalloproteinase-1 secretion. Additionally, LRG1 promoted SMAD2/SMAD3 phosphorylation in a pattern similar to that of TGF-β1 treatment. An inhibitor of TGF-β receptor 1 abolished LRG1-induced SMAD2 phosphorylation. RNA sequencing identified “extracellular region”, “extracellular space”, and “extracellular matrix” as the main Gene Ontology terms in the differentially expressed genes of fibroblasts treated with or without LRG1. LRG1 increased TGF-β1 mRNA levels, suggesting that LRG1 partially transactivates the expression of TGF-β1. Furthermore, an increased expression of type I collagen was also observed in fibroblasts grown in three-dimensional cultures on a collagen gel mimicking the dermis. LRG1 mRNA and protein levels were significantly reduced in elderly human skin tissues with weakened ECM integrity compared to in young human skin tissues. Taken together, our results suggest that LRG1 could retard skin aging by activating the TGF-β signaling pathway, increasing ECM deposition while decreasing its degradation. Full article

Prolidase is an enzyme hydrolyzing dipeptides containing proline or hydroxyprolineat the C-terminus and plays an important role in collagen turnover. Human prolidase is active as a dimer with the C-terminal domain containing two Mn²⁺ ions in its active site. The study aimed to develop a highly efficient expression system of recombinant human prolidase (rhPEPD) and to evaluate the effect of the N-terminal His-Tag on its enzymatic and biological activity. An optimized bacterial expression system and an optimized purification procedure for rhPEPD included the two-step rhPEPD purification procedure based on (i) affinity chromatography on an Ni²⁺ ion-bound chromatography column and (ii) gel filtration with the possibility of tag removal by selective digestion with protease Xa. As the study showed, a high concentration of IPTGand high temperature of induction led to a fast stimulation of gene expression, which as a result forced the host into an intensive and fast production of rhPEPD. The results demonstrated that a slow induction of gene expression (low concentration of inducing factor, temperature, and longer induction time) led to efficient protein production in the soluble fraction. Moreover, the study proved that the presence of His-Tag changed neither the expression pattern of EGFR-downstream signaling proteins nor the prolidase catalytic activity. Full article

The use of 3D in vitro tumor models has become a common trend in cancer biology studies as well as drug screening and preclinical testing of drug candidates. The transition from 2D to 3D matrix-based cell cultures requires modification of methods for assessing tumor growth. We propose the method for assessing the growth of tumor cells in a collagen hydrogel using macro-scale registration and quantification of the gel epi-fluorescence. The technique does not require gel destruction, can be used for real-time observation of fast (in seconds) cellular responses and demonstrates high agreement with cell counting approaches or measuring total DNA content. The potency of the method was proven in experiments aimed at testing cytotoxic activity of chemotherapeutic drug (cisplatin) and recombinant targeted toxin (DARPin-LoPE) against two different tumor cell lines genetically labelled with fluorescent proteins. Moreover, using fluorescent proteins with sensor properties allows registration of dynamic changes in cells’ metabolism, which was shown for the case of sensor of caspase 3 activity. Full article

Spheroids reproduce the tissue structure that is found in vivo more accurately than classic two-dimensional (2D) monolayer cultures. We cultured human periodontal ligament stem cells (HPLSCs) as spheroids that were embedded in collagen gel to examine whether their cementogenic differentiation could be enhanced by treatment with recombinant human plasminogen activator inhibitor-1 (rhPAI-1). The upregulated expression of cementum protein 1 (CEMP1) and cementum attachment protein (CAP), established cementoblast markers, was observed in the 2D monolayer HPLSCs that were treated with rhPAI-1 for 3 weeks compared with that in the control and osteogenic-induction medium groups. In the embedded HPLSC spheroids, rhPAI-1 treatment induced interplay between the spheroids and collagenous extracellular matrix (ECM), indicating that disaggregated HPLSCs migrated and spread into the surrounding ECM 72 h after three-dimensional (3D) culture. Western blot and immunocytochemistry analyses showed that the CEMP1 expression levels were significantly upregulated in the rhPAI-1-treated embedded HPLSC spheroids compared with all the 2D monolayer HPLSCs groups and the 3D spheroid groups. Therefore, 3D collagen-embedded spheroid culture in combination with rhPAI-1 treatment may be useful for facilitating cementogenic differentiation of HPLSCs. Full article

Systemic sclerosis (SSc; scleroderma) is characterized by life-threatening progressive multiorgan fibrosis orchestrated by profibrotic myofibroblasts originating from different sources. Because recent data demonstrated that the majority of myofibroblasts in a murine scleroderma model arise from adipocytic progenitors through the adipocyte-myofibroblast transition process, we sought to determine whether the SSc microenvironment may affect the differentiation potential of adipose-derived stem cells (ADSC). Normal human ADSC from three donors were treated with serum from SSc patients (n = 6), serum from healthy individuals (n = 6), or recombinant human transforming growth factor-β1 (TGFβ1) as positive control of myofibroblastic phenotype induction. ADSC were subjected to in vitro adipogenic differentiation for up to 21 days in the presence of different stimuli followed by lipid content quantification. In selected experiments, adipocytic and mesenchymal/myofibroblast marker gene and protein expression levels were assessed by Real-Time PCR, immunoblotting and immunofluorescence after administration of different stimuli for 72 and 96 h, respectively. Cell contractile phenotype was assayed by collagen gel contraction assay. Likewise stimulation with TGFβ1, SSc serum was able to significantly inhibit the adipocyte differentiation of ADSC as testified by a strong decrease in red-colored lipid droplets after 21 days of adipogenic induction. Treatment of ADSC either with SSc serum or TGFβ1 resulted in the acquisition of a myofibroblast-like phenotype characterized by a reduced expression of the adipocytic markers perilipin and adiponectin, a significant upregulation of the mesenchymal/myofibroblast markers α-SMA+ stress fibers, S100A4 and type I collagen, and an ability to effectively contract collagen gels. In SSc, the pathologic environment may favor the differentiation of ADSC into profibrotic and contractile myofibroblast-like cells. These findings strengthen the notion that the generation of myofibroblasts from ADSC may be relevant in SSc pathophysiology potentially representing a new target for the prevention/treatment of multiorgan fibrosis. Full article

Pancreatic ductal adenocarcinoma (PDA) is characterized by a pronounced fibroinflammatory stromal reaction consisting of inordinate levels of hyaluronan (HA), collagen, immune cells, and activated fibroblasts that work in concert to generate a robust physical barrier to the perfusion and diffusion of small molecule therapeutics. The targeted depletion of hyaluronan with a PEGylated recombinant human hyaluronidase (PEGPH20) lowers interstitial gel–fluid pressures and re-expands collapsed intratumoral vasculature, improving the delivery of concurrently administered agents. Here we report a non-invasive means of assessing biophysical responses to stromal intervention with quantitative multiparametric magnetic resonance imaging (MRI) at 14 Tesla (T). We found that spin-spin relaxation time T2 values and glycosaminoglycan chemical exchange saturation transfer (GagCEST) values decreased at 24 h, reflecting depletion of intratumoral HA content, and that these parameters recovered at 7 days concurrent with replenishment of intratumoral HA. This was also reflected in an increase in low-b apparent diffusion coefficient (ADC) at 24 h, consistent with improved tumor perfusion that again normalized at 7 days after treatment. Phantom imaging suggests that the GagCEST signal is driven by changes in HA versus other glycosaminoglycans. Thus, multiparametric magnetic resonance imaging (MRI) can be used as a non-invasive tool to assess therapeutic responses to targeted stromal therapy in PDA and likely other stroma-rich solid tumors that have high levels of hyaluronan and collagen. Full article

Transforming growth factor-beta (TGF-β) is involved in processes related to the differentiation and maturation of osteoprogenitor cells into osteoblasts. Rat bone marrow (BM) cells were cultured in a collagen-gel containing 0.5% fetal bovine serum (FBS) for 10 days in the presence of rhTGF (recombinant human TGF)-β1-F2, a fusion protein engineered to include a high-affinity collagen-binding decapeptide derived from von Willebrand factor. Subsequently, cells were moderately expanded in medium with 10% FBS for 4 days and treated with a short pulse of rhBMP (recombinant human bone morphogenetic protein)-2 for 4 h. During the last 2 days, dexamethasone and β-glycerophosphate were added to potentiate osteoinduction. Concomitant with an up-regulation of cell proliferation, DNA synthesis levels were determined. Polymerase chain reaction was performed to reveal the possible stemness of these cells. Osteogenic differentiation was evaluated in terms of alkaline phosphatase activity and mineralized matrix formation as well as by mRNA expression of osteogenic marker genes. Moreover, cells were placed inside diffusion chambers and implanted subcutaneously into the backs of adult rats for 4 weeks. Histological study provided evidence of cartilage and bone-like tissue formation. This experimental procedure is capable of selecting cell populations from BM that, in the presence of rhTGF-β1-F2 and rhBMP-2, achieve skeletogenic potential in vitro and in vivo. Full article