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Purpose: To compare bone substitutes composed of glycated collagen with synthetic micro-sized (1 to 10 µm) hydroxyapatite (OB) vs non-cross-linked collagen matrix with large-particle (250 to 1,000 µm) bovine-derived hydroxyapatite (BOC). Materials and Methods: The P1 to P4 premolars were bilaterally extracted from the mandibles of 19 Beagle dogs. After 21 days, osteotomies were created in each dog that received OB or BOC and were covered with a collagen membrane or were left untreated. The animals were randomly divided into three groups based on sacrifice time (4, 12, or 24 weeks). The right and left hemimandibles were trimmed to facilitate imaging and histology, and all tissues were placed in 10% neutral-buffered formalin. Microcomputed tomography (MicroCT 40 Scanner, Scanco) was used to analyze bone sections. Bone volume, residual material volume, and bone mineral density were determined for each treatment site (OB and BOC) based on a volume of interest that encompassed the original defect. Additionally, blinded histopathologic assessment (based on the ISO 10993-6 scoring system) and histomorphometry were performed on sections ground to < 100 µm thick and stained with Stevenel's blue. Results: No clinical side effects were noted. No statistical differences were observed for OB vs BOC regarding the mineral volume percentage. Compared to OB, BOC had significantly higher mean mineralization densities at 12 weeks (P < .01), but this difference did not extend to 24 weeks. For residual grafting material, bone maturation, alveolar ridge restoration, and inflammatory response, OB showed a residual amount of bone graft and no statistical differences compared to BOC. Conclusion: Both OB and BOC represent valid treatment options for critically sized bone defects. Both bone fillers outperformed the sham-operated, ungrafted (empty) control, demonstrating statistically improved bone growth and ridge restoration. [ABSTRACT FROM AUTHOR]

This study aimed to evaluate the properties of radiation cross‐linked collagen scaffold (RCS) and its efficacy for alveolar ridge preservation (ARP). RCS was prepared from collagen dispersion by electron beam irradiation and freeze‐drying. The microstructure, swelling ratio, area alteration and mechanical properties of RCS were characterized. Fifty‐four New Zealand rabbits performing incisor extraction on maxilla and mandible were randomly assigned into positive, sham operation or treatment groups. Micro‐computed tomography (micro‐CT) scans, performed after 1, 4, and 12 weeks of surgery, were to assess changes in ridge height at buccal and palatal side, in ridge width and in micromorphological parameters. Histological analysis accessed socket microarchitecture. The results showed that RCS had stable mechanical properties and morphologic features that provided a reliable physical support for ARP. Dimensional changes in treatment group revealed significantly greater vertical height at buccal (5.32 [3.37, 7.26] mm, p <.0001) and palatal (4.37 [2.66, 6.09] mm, p <.0001) side, and horizontal width at the maxilla (0.16 [0.04, 0.28] mm, p <.01) and mandible (0.33 [0.11, 0.54] mm, p <.01) than those in sham operation group after 12 weeks. The treatment group had advantage than positive group in vertical height preservation, quantitatively. The order and density of bone trabeculae were improved in treatment group. These findings indicated that RCS had the potential to serve as an effective scaffold for ARP. [ABSTRACT FROM AUTHOR]

BACKGROUND: The in vitro construction of intestinal smooth muscle layer, as an important component of the intestinal wall, has attracted much attention in the bionic construction of tissue-engineered intestinal canal. OBJECTIVE: To explore the effects of cyclic mechanical stretching on the growth activity of intestinal smooth muscle cells and the expression of functional genes within collagen strips. METHODS: The collagen band culture system of intestinal smooth muscle cells was constructed using a self-designed collagen strip stretching culture device with self-made rat tail collagen as a scaffold and primary rat intestinal smooth muscle cells as seed cells. EthD-1/Calcein-AM cell activity staining, magenta staining, cytoskeleton-Ki67 immunofluorescence staining were used to observe the growth activity and proliferation of the cells, and quantitative RT-PCR was used to detect the expression of desmin, α-sma, and vimentin functional genes. RESULTS AND CONCLUSION: The collagen band culture system of intestinal smooth muscle cells was successfully constructed, and intestinal smooth muscle cells in the band had good cell activity. The number of Ki67 positive cells increased and desmin, α-sma and vimentin were significantly overexpressed under cyclic stretching and dynamic culture conditions (P < 0.001). To conclude, mechanical stimulation is beneficial to maintain the growth phenotype of smooth muscle cells and promote their functional differentiation during three-dimensional culture in vitro. [ABSTRACT FROM AUTHOR]

Applied to the epicardium in-vivo , regenerative cardiac patches support the ventricular wall, reduce wall stresses, encourage ventricular wall thickening, and improve ventricular function. Scaffold engraftment, however, remains a challenge. After implantation, scaffolds are subject to the complex, time-varying, biomechanical environment of the myocardium. The mechanical capacity of engineered tissue to biomimetically deform and simultaneously support the damaged native tissue is crucial for its efficacy. To date, however, the biomechanical response of engineered tissue applied directly to live myocardium has not been characterized. In this paper, we utilize optical imaging of a Langendorff ex-vivo cardiac model to characterize the native deformation of the epicardium as well as that of attached engineered scaffolds. We utilize digital image correlation, linear strain, and 2D principal strain analysis to assess the mechanical compliance of acellular ice templated collagen scaffolds. Scaffolds had either aligned or isotropic porous architecture and were adhered directly to the live epicardial surface with either sutures or cyanoacrylate glue. We demonstrate that the biomechanical characteristics of native myocardial deformation on the epicardial surface can be reproduced by an ex-vivo cardiac model. Furthermore, we identified that scaffolds with unidirectionally aligned pores adhered with suture fixation most accurately recapitulated the deformation of the native epicardium. Our study contributes a translational characterization methodology to assess the physio-mechanical performance of engineered cardiac tissue and adds to the growing body of evidence showing that anisotropic scaffold architecture improves the functional biomimetic capacity of engineered cardiac tissue. Engineered cardiac tissue offers potential for myocardial repair, but engraftment remains a challenge. In-vivo , engineered scaffolds are subject to complex biomechanical stresses and the mechanical capacity of scaffolds to biomimetically deform is critical. To date, the biomechanical response of engineered scaffolds applied to live myocardium has not been characterized. In this paper, we utilize optical imaging of an ex-vivo cardiac model to characterize the deformation of the native epicardium and scaffolds attached directly to the heart. Comparing scaffold architecture and fixation method, we demonstrate that sutured scaffolds with anisotropic pores aligned with the native alignment of the superficial myocardium best recapitulate native deformation. Our study contributes a physio-mechanical characterization methodology for cardiac tissue engineering scaffolds. [Display omitted] [ABSTRACT FROM AUTHOR]

Tissues and organs contain an extracellular matrix (ECM). In the case of blood vessels, endothelium cells are anchored to a specialized basement membrane (BM) embedded inside the interstitial matrix (IM). We introduce a multi-structural collagen-based scaffold with embedded microchannels that mimics in vivo structures within vessels. Our scaffold consists of two parts, each containing two collagen layers, i.e., a 3D porous collagen layer analogous to IM lined with a thin 2D collagen film resembling the BM. Enclosed microchannels were fabricated using contact microprinting. Microchannel test structures with different sizes ranging from 300 to 800 µm were examined for their fabrication reproducibility. The heights and perimeters of the fabricated microchannels were ~20% less than their corresponding values in the replication PDMS mold; however, microchannel widths were significantly closer to their replica dimensions. The stiffness, permeability, and pore size properties of the 2D and 3D collagen layers were measured. The permeability of the 2D collagen film was negligible, making it suitable for mimicking the BM of large blood vessels. A leakage test at various volumetric flow rates applied to the microchannels showed no discharge, thereby verifying the reliability of the proposed integrated 2D/3D collagen parts and the contact printing method used for bonding them in the scaffold. In the future, multi-cell culturing will be performed within the 3D porous collagen and against the 2D membrane inside the microchannel, hence preparing this scaffold for studying a variety of blood vessel–tissue interfaces. Also, thicker collagen scaffold tissues will be fabricated by stacking several layers of the proposed scaffold. [ABSTRACT FROM AUTHOR]

Southern flounder skin pigmentation is a critical phenotypic characteristic for this species' survival in the natural environment. Normal pigmentation allows rapid changes of color for concealment to capture prey and UV light protection. In contrast, highly visible hypopigmented pseudo-albinos exhibit a compromised immune system and are vulnerable to predation, sensitive to UV exposure, and likely have poor survival in the wild. Skin and brain tissue samples from normally pigmented and hypopigmented individuals were analyzed with next-generation RNA sequencing. A total of 1,589,613 transcripts were used to identify 952,825 genes to assemble a de novo transcriptome, with 99.43% of genes mapped to the assembly. Differential gene expression and gene enrichment analysis of contrasting tissues and phenotypes revealed that pseudo-albino individuals appeared more susceptible to environmental stress, UV light exposure, hypoxia, and osmotic stress. The pseudo-albinos' restricted immune response showed upregulated genes linked to cancer development, signaling and response, skin tissue formation, regeneration, and healing. The data indicate that a modified skin collagen structure likely affects melanocyte differentiation and distribution, generating the pseudo-albino phenotype. In addition, the comparison of the brain transcriptome revealed changes in myelination and melanocyte stem cell activity, which may indicate modified brain function, reduced melanocyte migration, and impaired vision. [ABSTRACT FROM AUTHOR]

Objectives: This randomized clinical trial focused on patients with thin peri-implant soft-tissue height (STH) (≤ 2.5 mm) and investigated the impact of an allogenic collagen scaffold (aCS) on supracrestal tissue height and marginal bone loss (MBL). Material & methods: Forty patients received bone level implants and were randomly assigned to the test group with simultaneous tissue thickening with aCS or the control group. After three months, prosthetic restoration occurred. STH measurements were taken at baseline (T0) and reopening surgery (TR), with MBL assessed at 12 months (T1). Descriptive statistics were calculated for continuous variables, and counts for categorical variables (significance level, p = 0.05). Results: At T1, 37 patients were available. At T0, control and test groups had mean STH values of 2.3 ± 0.3 mm and 2.1 ± 0.4 mm. TR revealed mean STH values of 2.3 ± 0.2 mm (control) and 2.6 ± 0.7 mm (test), with a significant tissue thickening of 0.5 ± 0.6 mm in the test group (p < 0.03). At T1, control and test groups showed MBL mean values of 1.1 ± 0.8 mm and 1.0 ± 0.6 mm, with a moderate but significant correlation with STH thickening (-0.34), implant position (0.43), history of periodontitis (0.39), and smoking status (0.27). Conclusion: The use of an aCS protocol resulted in soft tissue thickening but did not reach a threshold to reliably reduce MBL compared to the control group within the study’s limitations. Clinical relevance: Peri-implant STH is crucial for maintaining peri-implant marginal bone stability. Marginal bone stability represents a crucial factor in prevention of peri-implantitis development. German register of clinical trial registration number DRKS00033290. [ABSTRACT FROM AUTHOR]

Ovarian cancer poses a significant threat to patients in its advanced stages, often with limited treatment options available. In such cases, palliative management becomes the primary approach to maintaining a reasonable quality of life. Therefore, the administration of any medication that can benefit patients without a curative option holds potential. Resveratrol, a natural compound known for its in vitro anticancer activities, has generated contrasting results in vivo and human studies. In this study, we aimed to assess the anticancer effects of resveratrol on ovarian cancer cells grown on the chorioallantoic membrane (CAM) of chicken embryos. Two ovarian cancer cell lines, OVCAR-8 and SKOV-3, were cultured in collagen scaffolds for four days before being implanted on the CAM of chicken embryos on day 7. Different doses of resveratrol were applied to the CAM every two days for six days. Subsequently, CAM tissues were excised, fixed, and subjected to histological analysis. Some CAM tumours were extracted to analyse proteins through Western blotting. Our findings indicate that specific doses of resveratrol significantly reduce angiogenic activities, pNF-κB levels, and SLUG protein levels by using immunohistochemistry. These results suggest that resveratrol may have the potential to impact the behaviour of ovarian cancer CAM tumours, thereby warranting further consideration as a complementary treatment option for women with incurable ovarian cancer. [ABSTRACT FROM AUTHOR]

Recombinant human bone morphogenetic protein 2 (rhBMP-2) is an FDA-approved growth factor for bone regeneration and repair in medical practice. The therapeutic effects of rhBMP-2 may be enhanced through specific binding to extracellular matrix (ECM)-like scaffolds. Here, we report the selection of a novel rhBMP-2-specific DNA aptamer, functionalization of the aptamer in an ECM-like scaffold, and its application in a cellular context. A DNA aptamer BA1 was evolved and shown to have high affinity and specificity to rhBMP-2. A molecular docking model demonstrated that BA1 was probably bound to rhBMP-2 at its heparin-binding domain, as verified with experimental competitive binding assays. The BA1 aptamer was used to functionalize a type I collagen scaffold, and fraction ratios were optimized to mimic the natural ECM. Studies in the myoblast cell model C2C12 showed that the aptamer-enhanced scaffold could specifically augment the osteo-inductive function of rhBMP-2 in vitro. This aptamer-functionalized scaffold may have value in enhancing rhBMP-2-mediated bone regeneration. [ABSTRACT FROM AUTHOR]

Peripheral nerve injuries (PNIs) include complete and partial transection, crushing, and chronic compression injuries. Hollow absorbable conduits are used to treat complete transection with short defects, while wrapping the injured part with an absorbent material promotes nerve recovery by inhibiting inflammatory cell infiltration and scar tissue formation in crush injuries. For treatment of partially transected nerve injuries (PTNIs), such as injection‐related iatrogenic PNI, whether wrapping the entire nerve, including the injury site, or bridging the transected fascicle with an artificial nerve conduit (ANC) is beneficial remains to be verified. The purpose of this study was to investigate whether wrapping the injured nerve and placing collagen fibers as scaffolds at the nerve defect site contribute to neural recovery in PTNI. A unilateral 5‐mm partial nerve defect was created at the mid‐thigh level in a rat sciatic nerve injury model. Fifty‐four Sprague–Dawley (SD) rats (150–250 g) were divided into three groups (n = 9 each): group 1, collagen fibers were placed in the nerve defect and the sciatic nerve was wrapped with collagen conduit; group 2, the sciatic nerve was wrapped by collagen conduit without collagen fibers; and group 3, nerve defect was reconstructed with collagen‐filled conduit. Nerve regeneration was evaluated by analyses of gait, electrophysiology, wet muscle weight, and axon numbers with immunohistochemistry at 12 and 24 weeks. Dorsiflexion angles among all groups improved significantly from 12 to 24 weeks postoperatively. At 24 weeks postoperatively, compound muscle action potential amplitudes (CMAPs) of tibialis anterior were 5.26 ± 4.64, 1.31 ± 1.17, and 0.14 ± 0.24 mV (p <.05), CMAPs of gastrocnemius were 21.3 ± 5.98, 15.4 ± 5.46, and 13.11 ± 3.91 mV in groups 1, 2, and 3, respectively; and the value of group 1 was significantly higher than that of group 3 (p <.05). Axon numbers were 2194 ± 629; 1106 ± 645; and 805 ± 907 in groups 1, 2, and 3, respectively (p <.05). For PTNI reconstruction, artificial nerve wrap (ANW) was superior to ANC. Providing collagen scaffold at the nerve defect site enhanced nerve recovery during reconstruction with ANW. [ABSTRACT FROM AUTHOR]

Background: Shoulder impingement syndrome (SIS) is one of the most common diseases of the shoulder and can be addressed with various therapeutic concepts. Orthobiological agents such as platelet rich plasma with a low side effect rate gain importance in the conservative treatment of SIS. Currently, the knowledge about success rate influencing factors, such as the growth factors (GF) concentration or acromion type, is limited. The aim of this study was to analyze the clinical outcome in the therapy of external SIS using autologous conditioned plasma combined with recombinant human collagen scaffold (ACP/STR) injection in comparison with a corticosteroid-local anesthetic (CSA) injection. Additionally, the influence of potential limiting factors such as GF concentration, age and acromial morphology was proved. Materials and methods: This prospective pseudo-randomized trial recruited 58 patients with external SIS who received an ultrasound-guided subacromial injection either an ACP/STR or a CSA followed by physical therapy. Follow-up (FU) was performed at 6 weeks, 3 and 6 months. The outcome was assessed with Constant–Murley score, disability of arm, shoulder and hand score and simple shoulder test. The concentration of GF was measured using ELISA. Results: During the FU, the improvement of outcome measures was observed with no differences between both groups. Shoulder force was significantly increased in the ACP/STR group (p < 0.01). We found no correlation between the amount of GF and age or gender in the ACP/STR patients. An acromion Bigliani type III predisposes for therapy failure (p < 0.001, OR = 56) in both treatment groups. Conclusions: Patients with SIS benefit regarding to PROMs after both ACP/STR and CSA injection and physical therapy. Patients who received ACP/STR obtained superior improvement in force. The quantity of GF did not vary depending on the age, so that ACP/STR can be a treatment option for SIS in elderly patients with multimorbidity. The presence of an acromion type III seems to be a predictive factor for limited effectivity of injections in the clinical management of SIS. [ABSTRACT FROM AUTHOR]

Tissues and organs contain an extracellular matrix (ECM). In the case of blood vessels, endothelium cells are anchored to a specialized basement membrane (BM) embedded inside the interstitial matrix (IM). We introduce a multi-structural collagen-based scaffold with embedded microchannels that mimics in vivo structures within vessels. Our scaffold consists of two parts, each containing two collagen layers, i.e., a 3D porous collagen layer analogous to IM lined with a thin 2D collagen film resembling the BM. Enclosed microchannels were fabricated using contact microprinting. Microchannel test structures with different sizes ranging from 300 to 800 µm were examined for their fabrication reproducibility. The heights and perimeters of the fabricated microchannels were ~20% less than their corresponding values in the replication PDMS mold; however, microchannel widths were significantly closer to their replica dimensions. The stiffness, permeability, and pore size properties of the 2D and 3D collagen layers were measured. The permeability of the 2D collagen film was negligible, making it suitable for mimicking the BM of large blood vessels. A leakage test at various volumetric flow rates applied to the microchannels showed no discharge, thereby verifying the reliability of the proposed integrated 2D/3D collagen parts and the contact printing method used for bonding them in the scaffold. In the future, multi-cell culturing will be performed within the 3D porous collagen and against the 2D membrane inside the microchannel, hence preparing this scaffold for studying a variety of blood vessel–tissue interfaces. Also, thicker collagen scaffold tissues will be fabricated by stacking several layers of the proposed scaffold.

Southern flounder skin pigmentation is a critical phenotypic characteristic for this species’ survival in the natural environment. Normal pigmentation allows rapid changes of color for concealment to capture prey and UV light protection. In contrast, highly visible hypopigmented pseudo-albinos exhibit a compromised immune system and are vulnerable to predation, sensitive to UV exposure, and likely have poor survival in the wild. Skin and brain tissue samples from normally pigmented and hypopigmented individuals were analyzed with next-generation RNA sequencing. A total of 1,589,613 transcripts were used to identify 952,825 genes to assemble a de novo transcriptome, with 99.43% of genes mapped to the assembly. Differential gene expression and gene enrichment analysis of contrasting tissues and phenotypes revealed that pseudo-albino individuals appeared more susceptible to environmental stress, UV light exposure, hypoxia, and osmotic stress. The pseudo-albinos’ restricted immune response showed upregulated genes linked to cancer development, signaling and response, skin tissue formation, regeneration, and healing. The data indicate that a modified skin collagen structure likely affects melanocyte differentiation and distribution, generating the pseudo-albino phenotype. In addition, the comparison of the brain transcriptome revealed changes in myelination and melanocyte stem cell activity, which may indicate modified brain function, reduced melanocyte migration, and impaired vision.

Mineralization can help improve the mechanical properties and degradation rates of collagen scaffolds while ensuring good biocompatibility, thereby providing a suitable microenvironment for osteogenic differentiation. Intrafibrillar/extrafibrillar mineralization of collagen is promoted by polymer‐induced liquid precursors (PILP). In this study, polyacrylamide (PAM) was introduced to PILP which synergistically mineralized collagen scaffold intrafibrillarly and extrafibrillarly. PAM and polyacrylic acid (PAA) can stabilize the amorphous calcium phosphate (ACP), to form a PILP of PAM and PAA/ACP. As a control, another scaffold material was formed using the conventional mineralization method, that is, soaking collagen in a simulated body fluid. Collagen mineralization was characterized using SEM and TEM. After 7 days of mineralization with PILP, intrafibrillar crystallization of collagen was significantly higher than that in the control group, and the stiffness and modulus of this scaffold material significantly increased. Cellular experiment results indicated that the PILP‐mineralized collagen scaffold was biocompatible and promoted the osteogenic differentiation of MC3T3‐E1 pre‐osteoblasts. Biomimetic mineralization by PILP will assist the fabrication of mineralized collagen scaffold for bone repair applications. [ABSTRACT FROM AUTHOR]

Recombinant human bone morphogenetic protein 2 (rhBMP-2) is an FDA-approved growth factor for bone regeneration and repair in medical practice. The therapeutic effects of rhBMP-2 may be enhanced through specific binding to extracellular matrix (ECM)-like scaffolds. Here, we report the selection of a novel rhBMP-2-specific DNA aptamer, functionalization of the aptamer in an ECM-like scaffold, and its application in a cellular context. A DNA aptamer BA1 was evolved and shown to have high affinity and specificity to rhBMP-2. A molecular docking model demonstrated that BA1 was probably bound to rhBMP-2 at its heparin-binding domain, as verified with experimental competitive binding assays. The BA1 aptamer was used to functionalize a type I collagen scaffold, and fraction ratios were optimized to mimic the natural ECM. Studies in the myoblast cell model C2C12 showed that the aptamer-enhanced scaffold could specifically augment the osteo-inductive function of rhBMP-2 in vitro. This aptamer-functionalized scaffold may have value in enhancing rhBMP-2-mediated bone regeneration.

Aquaculture is the fast-growing industry and, consequently, fish waste management is becoming a rising concern. Fish scale trash contains collagen as a biomaterial which offers excellent properties such as biocompatibility, biodegradability, porosity, low toxicity, cell adhesion and water absorption capacity which makes it suitable for biomedical research in the form of scaffolds. But the scaffolds have the limitation of having less stability. To overcome this shortcoming, collagen scaffolds can either be modified by chemical or physical treatments such as crosslinking with suitable polymers and dehydrothermal treatment, respectively. In the present study, extraction of fish collagen from the scales of Cyprinus carpio was performed followed by crosslinking with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) & N-hydroxysuccinimide (NHS). Comparative characterization was done by techniques such as ATR-FTIR, FESEM and TGA to analyse the chemical composition, surface morphology and thermal degradation of fish collagen scaffold (CS) with EDC/NHS crosslinked collagen scaffold (EDC/NHS-CS). Crosslinked collagen scaffold's antimicrobial activity was also determined with agar well diffusion assay by using S. aureus strain of bacteria and cytotoxicity on mouse fibroblast cells (NIH/3T3) by MTT assay. Moreover, the biomedical industry wants to shift to sustainable approaches and fish collagen-based scaffolds may prove to be a potential source in tissue engineering. And thus, also help in the management of fish waste. [ABSTRACT FROM AUTHOR]

Excessive tissue damage or loss has been solved by guided tissue regeneration and guided bone regeneration theories. However, the unfavorable degradation property of the resorbable collagen scaffold brings a big challenge to support soft tissue stabilization and time‐consuming osteogenesis. The combined effect for soft tissue and bone of the collagen scaffold with better degradation pattern has not been clearly proven. This study determined whether the double surfaces of crosslinked collagen scaffolds could optimize the combined soft tissue repair and osteogenesis. In this study, we applied the chemically crosslinking treatment to the commercially available collagen scaffolds. Surface characterization, mechanical property and cell proliferation in vitro were evaluated. Combined bilateral skin and bone defects were established with the smooth surface of scaffold facing the skin defect and the rough surface facing the bone defect on the calvaria of rat. Micro‐CT and histological evaluation were applied to determine the scaffold degradation pattern, soft tissue repair and osteogenesis. The crosslinked collagen scaffolds showed comparably favorable surface porosity, structure intactness, superhydrophilicity and mechanical properties. Compared to the native scaffolds, the crosslinked scaffolds could optimize the combined soft tissue repair and osteogenesis by preferably prolonged degradation time. Early pro‐angiogenesis facilitated soft tissue repair and osteogenesis by upregulated soft tissue matrix degradation and balanced pro‐osteogenesis with limited osteoclast‐mediated bone resorption. Taken together, this study offers a promising repair strategy for the combined soft tissue and bone defects. Further, the possible mechanism of controllable scaffold degradation should be conducted. [ABSTRACT FROM AUTHOR]

Hyaline cartilage has very limited repair capability and cannot be rebuilt predictably using conventional treatments. This study presents Autologous Chondrocyte Implantation (ACI) on two different scaffolds for the treatment of lesions in hyaline cartilage in rabbits. The first one is a commercially available scaffold (Chondro–Gide) made of collagen type I/III and the second one is a polyethersulfone (PES) synthetic membrane, manufactured by phase inversion. The revolutionary idea in the present study is the fact that we used PES membranes, which have unique features and benefits that are desirable for the 3D cultivation of chondrocytes. Sixty-four White New Zealand rabbits were used in this research. Defects penetrating into the subchondral bone were filled with or without the placement of chondrocytes on collagen or PES membranes after two weeks of culture. The expression of the gene encoding type II procollagen, a molecular marker of chondrocytes, was evaluated. Elemental analysis was performed to estimate the weight of tissue grown on the PES membrane. The reparative tissue was analyzed macroscopically and histologically after surgery at 12, 25, and 52 weeks. RT-PCR analysis of the mRNA isolated from cells detached from the polysulphonic membrane revealed the expression of type II procollagen. The elementary analysis of polysulphonic membrane slices after 2 weeks of culture with chondrocytes revealed a concentration of 0.23 mg of tissue on one part of the membrane. Macroscopic and microscopic evaluation indicated that the quality of regenerated tissue was similar after the transplantation of cells placed on polysulphonic or collagen membranes. The established method for the culture and transplantation of chondrocytes placed on polysulphonic membranes resulted in the growth of the regenerated tissue, revealing the morphology of hyaline-like cartilage to be of similar quality to collagen membranes. [ABSTRACT FROM AUTHOR]

The combination of scaffolds with recombinant human epidermal growth factor (rhEGF) protein can enhance defective bone healing via synergistic activation to stimulate cellular growth, differentiation, and survival. We examined the biopotentials of an rhEGF-loaded absorbable collagen scaffold (ACS) using a mouse model of calvarial defects, in which the rhEGF was produced from a plant cell suspension culture system because of several systemic advantages. Here, we showed a successful and large-scale production of plant-cell-derived rhEGF protein (p-rhEGF) by introducing an expression vector that cloned with its cDNA under the control of rice α-amylase 3D promoter into rice calli (Oryza sativa L. cv. Dongjin). Implantation with p-rhEGF (5 μg)-loaded ACSs into critical-sized calvarial defects enhanced new bone formation and the expression of osteoblast-specific markers in the defected regions greater than implantation with ACSs alone did. The potency of p-rhEGF-induced bone healing was comparable with that of Escherichia coli-derived rhEGF protein. The exogenous addition of p-rhEGF increased the proliferation of human periodontal ligament cells and augmented the induction of interleukin 8, bone morphogenetic protein 2, and vascular endothelial growth factor in the cells. Collectively, this study demonstrates the successful and convenient production of p-rhEGF, as well as its potency to enhance ACS-mediated bone regeneration by activating cellular responses that are required for wound healing. [ABSTRACT FROM AUTHOR]

Case: A 16-year-old female competitive gymnast presented to our orthopaedic clinic with an acute Achilles tendon rupture at the myotendinous junction. Direct end-to-end repair was performed and augmented with a bioinductive collagen patch. The patient had increased tendon thickness at 6 months postoperatively, as well as significant improvements in strength and range of motion at 12 months. Conclusion: Bioinductive collagen patch augmentation of Achilles tendon repair may be a useful adjunct for myotendinous junction Achilles ruptures, particularly in high-demand patients including competitive gymnasts. [ABSTRACT FROM AUTHOR]

The process of generating type I/II collagen scaffolds is fraught with bubble formation, which can interfere with the three-dimensional structure of the scaffold. Herein, we applied low-temperature vacuum freeze-drying to remove mixed air bubbles under negative pressure. Type I and II rubber sponges were acid-solubilized via acid lysis and enzymolysis. Thereafter, vacuum negative pressure was applied to remove bubbles, and the cover glass press method was applied to shape the type I/II original scaffold. Vacuum negative pressure was applied for a second time to remove any residual bubbles. Subsequent application of carbamide/N-hydroxysuccinimide cross-linked the scaffold. The traditional method was used as the control group. The structure and number of residual bubbles and pore sizes of the two scaffolds were compared. Based on the relationship between the pressure and the number of residual bubbles, a curve was created, and the time of ice formation was calculated. The bubble content of the experimental group was significantly lower than that of the control group (P < 0.05). The pore diameter of the type I/II collagen scaffold was higher in the experimental group than in the control group. The time of icing effect of type I and II collagen solution was 136.54 ± 5.26 and 144.40 ± 6.45 s, respectively. The experimental scaffold had a more regular structure with actively proliferating chondrocytes that possessed adherent pseudopodia. The findings indicated that the vacuum negative pressure method did not affect the physical or chemical properties of collagen, and these scaffolds exhibited good biocompatibility with chondrocytes. [ABSTRACT FROM AUTHOR]

The volume of the bone in a site past an extraction degrades significantly and thus it is imperative to evaluate the situation for implant placement. Besides the need for sufficient bone, the amount and quality of the soft tissue covering the bone in the missing tooth area and nature of the adjacent teeth must be carefully assessed. In anterior sites, reconstructive surgery is usually performed to restore these hard and soft tissues, mainly for esthetic reasons, but it is equally essential in posterior sites to ensure adequate functional support. In guided bone regeneration procedures, barrier membranes block the augmented areas, provide and maintain space for regenerative material, and protect the blood clot, allowing a normal wound stability process. Clinicians prefer using resorbable membranes in most cases, whereas a nonresorbable membrane is selected to correct large defects. This report proposes the use of a collagen scaffold as a core material for guided bone regeneration in the case of a missing tooth between two existing teeth, when there is sufficient bone to place an implant but a horizontal defect is present in the crestal ridge. The tested question is whether a thick, reinforced, resorbable collagen scaffold (Ossix Volumax) can provide a stable basis for restoring the lost volume of a deficient ridge. The regeneration procedure presented with the collagen scaffold resulted in restoration of the lost tissue volume and a favorable lifelike emergence profile for the implant-supported crown. This augmentation procedure is simpler to perform in certain cases than existing procedures with bone substitute material and/or an interpositional connective tissue graft harvested from a remote donor site, the harvest of which is not required. [ABSTRACT FROM AUTHOR]

Background: Single-step all-arthroscopic techniques have gained popularity recently in the treatment of osteochondral lesions of the talus (OLT). Concomitant subchondral bone defects led surgeons to add autologous bone grafting to the surgical procedures. Collagen scaffolds have been used widely for stabilization of the reconstruction and regeneration of the articular surface. Purpose: To compare single-step all-arthroscopic treatment of OLT consisting of debridement, microfracture, autologous bone grafting, and application of fibrin sealant in 2 patient groups: with versus without collagen scaffold. Study Design: Cohort study; Level of evidence, 3. Methods: Included were 94 patients who underwent single-step all-arthroscopic treatment for OLT. Autologous bone grafting was applied to 48 patients (BG group), while autologous bone grafting plus collagen scaffold was applied to 46 patients (BG+S group). A fibrin sealant was applied to both groups. Clinical outcomes were assessed with the American Orthopaedic Foot & Ankle Society (AOFAS) score and the visual analog scale (VAS) for pain. Radiological outcomes were evaluated with the magnetic resonance observation of cartilage repair tissue score. The mean follow-up time was 69.3 ± 20.7 months. Results: Patients in both groups showed statistically significant improvement in pre- to postoperative AOFAS and VAS scores (P <.001 for all), with no difference between groups in AOFAS and VAS score improvement. Complete healing with or without hypertrophy was achieved in 42 patients in the BG group (87.5%) and 38 patients in the BG+S group (82.6%). Conclusion: The treatment of bone lesions in OLT may be the ultimate goal to obtain successful outcomes, in which case using a collagen scaffold besides grafting may not affect clinical and radiological outcomes. [ABSTRACT FROM AUTHOR]

Severe endometrium damage causes pathological conditions such as thin endometrium and intrauterine adhesion, resulting in uterine factor infertility. Mesenchymal stem cell (MSC) therapy is a promising strategy in endometrial repair; yet, exogenous MSCs still raise concerns for safety and ethical issues. Human adipose-derived mesenchymal stem cells (ADMSCs) residing in adipose tissue have high translational potentials due to their autologous origin. To harness the high translation potentials of ADMSC in clinical endometrium regeneration, here we constructed an ADMSCs composited porous scaffold (CS/ADMSC) and evaluated its effectiveness on endometrial regeneration in a rat endometrium-injury model. We found that CS/ADMSC intrauterine implantation (i) promoted endometrial thickness and gland number, (ii) enhanced tissue angiogenesis, (iii) reduced fibrosis and (iv) restored fertility. We ascertained the pro-proliferation, pro-angiogenesis, immunomodulating and anti-fibrotic effects of CS/ADMSC in vitro and revealed that the CS/ADMSC influenced extracellular matrix composition and organization by a transcriptomic analysis. Our results demonstrated the effectiveness of CS/ADMSC for endometrial regeneration and provided solid proof for our future clinical study. [ABSTRACT FROM AUTHOR]

Microglia activation after spinal cord injury (SCI) is a double-edged sword, modulation of the activated microglia populations toward pro-regenerative phenotypes highlights the potential therapeutic implications. P2Y12, a microglia-specific marker, remains underexplored in its capacity to polarize microglial activation populations in SCI repair. We aimed to explore the effects of modulating P2Y12 on microglia function after spinal cord injury, and further on axonal regeneration and motor recovery after spinal cord injury. The study employed both in vitro and in vivo models, using BV2 cells and a mouse model of SCI, respectively. Ticagrelor, a P2Y12 antagonist, was administered via a collagen scaffold to ensure stable and sustained release. Transcriptome sequencing analysis, immunofluorescence staining, and Basso Mouse Scale (BMS) scores were used to assess microglial activation, axonal regeneration, and functional recovery. Herein, we observed P2Y12+ microglia localized predominantly at the lesion periphery within 3 days post injury (dpi), manifesting a pro-inflammatory phenotype, but not anti-inflammatory phenotype. In vitro investigations revealed that P2Y12 inhibition of the activated microglia curtailed pro-inflammatory differentiation while augmenting anti-inflammatory differentiation. Leveraging this insight, we engineered a collagen scaffold-based delivery system for sustained release of the P2Y12 antagonist, ticagrelor, at the injury site in a mouse complete SCI model. Notably, P2Y12 suppression markedly enhanced axonal regeneration within the injured site and ameliorated lower limb motor functions in SCI mice. Collectively, our findings illuminate P2Y12-targeted microglial modulation as a promising therapeutic approach for SCI. [Display omitted] [ABSTRACT FROM AUTHOR]

Faithful modeling of tissues and organs requires the development of systems reflecting their dynamic 3D cellular architecture and organization. Current technologies suffer from a lack of design flexibility and complex prototyping, preventing their broad adoption by the scientific community. To make 3D cell culture more available and adaptable we here describe the use of the fused deposition modeling (FDM) technology to rapid-prototype 3D printed perfusion bioreactors. Our 3D printed bioreactors are made of polylactic acid resulting in reusable systems customizable in size and shape. Following design confirmation, our bioreactors were biologically validated for the culture of human mesenchymal stromal cells under perfusion for up to 2 weeks on collagen scaffolds. Microenvironments of various size/volume (6–12 mm in diameter) could be engineered, by modulating the 3D printed bioreactor design. Metabolic assay and confocal microscopy confirmed the homogenous mesenchymal cell distribution throughout the material pores. The resulting human microenvironments were further exploited for the maintenance of human hematopoietic stem cells. Following 1 week of stromal coculture, we report the recapitulation of 3D interactions between the mesenchymal and hematopoietic fractions, associated with a phenotypic expansion of the blood stem cell populations.Our data confirm that perfusion bioreactors fit for cell culture can be generated using a 3D printing technology and exploited for the 3D modeling of complex stem cell systems. Our approach opens the gates for a more faithful investigation of cellular processes in relation to a dynamic 3D microenvironment.

The conjunctiva covers the largest area of ocular surface and is responsible for tear balance and clear vision. After trauma or surgery, the conjunctiva is prone to scarring and contracture. Transplantation with suture often implies numerous complications, such as inflammation, suture erosion, granuloma. And the suture needs to be removed, which means a secondary trauma. In this study, a bioadhesive hydrogel (GMO) for sutureless conjunctival transplantation was developed based on a semi-interpenetrating polymer network (sIPN) consisting of gelatin methacrylate (GelMA) and oxidized hyaluronic acid (OHA). The maximum adhesion strength was 157 ± 17 kPa, and the burst pressure was 357 ± 29 kPa, which was 15 times higher than the human intraocular pressure (IOP). GMO bioadhesive hydrogel significantly improved surgical efficiency and secured the collagen scaffold firmly to a rabbit conjunctival defect. The sutureless transplantation approach revealed the promoted tissue repair without scar. In conclusion, GMO bioadhesive may be an attractive alternative to suture for ocular surface reconstruction by avoiding suture-related complications and improving clinical outcome. Conjunctival tissue is prone to scarring and contracture after trauma, and surgery with sutures often implies numerous complications. In this study, the ocular surface reconstruction was achieved by sutureless transplantation of conjunctival scaffold using bioadhesive hydrogel. The prepared GMO bioadhesive based on the semi-interpenetrating network of gelatin methacrylate (GelMA) and oxidized hyaluronic acid (OHA) had favorable adhesion and mechanical properties. The sutureless transplantation approach significantly improved the operation efficiency, avoided suture-related complications, and promoted the regeneration of conjunctiva. This study highlights the great potential of the sutureless repair strategy for clinical application in ocular surface reconstruction. [Display omitted] [ABSTRACT FROM AUTHOR]

The architectural and physiomechanical properties of regenerative scaffolds have been shown to improve engineered tissue function at both a cellular and tissue level. The fabrication of regenerative three-dimensional scaffolds that precisely replicate the complex hierarchical structure of native tissue, however, remains a challenge. The aim of this work is therefore two-fold: i) demonstrate an innovative multidirectional freeze-casting system to afford precise architectural control of ice-templated collagen scaffolds; and ii) present a predictive simulation as an experimental design tool for bespoke scaffold architecture. We used embedded heat sources within the freeze-casting mold to manipulate the local thermal environment during solidification of ice-templated collagen scaffolds. The resultant scaffolds comprised complex and spatially varied lamellar orientations that correlated with the imposed thermal environment and could be readily controlled by varying the geometry and power of the heat sources. The complex macro-architecture did not interrupt the hierarchical features characteristic of ice-templated scaffolds, but pore orientation had a significant impact on the stiffness of resultant structures under compression. Furthermore, our finite element model (FEM) accurately predicted the thermal environment and illustrated the freezing front topography within the mold during solidification. The lamellar orientation of freeze-cast scaffolds was also predicted using thermal gradient vector direction immediately prior to phase change. In combination our FEM and bespoke freeze-casting system present an exciting opportunity for tailored architectural design of ice-templated regenerative scaffolds that mimic the complex hierarchical environment of the native extracellular matrix. Biomimetic scaffold structure improves engineered tissue function, but the fabrication of three-dimensional scaffolds that precisely replicate the complex hierarchical structure of native tissue remains a challenge. Here, we leverage the robust relationship between thermal gradients and lamellar orientation of ice-templated collagen scaffolds to develop a multidirectional freeze-casting system with precise control of the thermal environment and consequently the complex lamellar structure of resultant scaffolds. Demonstrating the diversity of our approach, we identify heat source geometry and power as control parameters for complex lamellar orientations. We simultaneously present a finite element model (FEM) that describes the three-dimensional thermal environment during solidification and accurately predicts lamellar structure of resultant scaffolds. The model serves as a design tool for bespoke regenerative scaffolds. [Display omitted] [ABSTRACT FROM AUTHOR]

Collagen is key component of extracellular matrix for human and animals. It can be extracted and applied for various field especially in tissue engineering as main component for wound healing. Collagen especially type I collagen is used in skin regeneration process due to its high compatibility with donor site and low antigenicity besides of having suitable properties for wound healing like high cell proliferation and adhesion. However, the usage of collagen alone would not give maximum wound healing since collagen degrades faster, has poor structure for a scaffold and attracts bacteria due to high moisture content. Therefore, to produce a good collagen scaffold, it must be incorporated with functional biomaterials to enhance the characteristic of the collagen and fabricated for a better scaffold structure. The objective of this systematic review is to summarize the relevant literature for in vitro study on collagen application in wound healing by focusing on the source of collagen, biomaterials and fabrication methods used in making collagen scaffold. Three databases were searched; PubMed, Scopus and Science Direct. Keywords used were: collagen, recombinant collagen, collagen scaffold, application and wound healing. A total of 1105 were articles screened but only 50 articles were suitable and were further reviewed. Collagen in wound healing study has versatility in terms of the source of collagen used, the biomaterials combined with the collagen to make an enhanced scaffold, and the fabrication methods to create a desirable structure of collagen scaffold. [ABSTRACT FROM AUTHOR]

The healing of severe chronic skin wounds in chronic diabetic patients is still a huge clinical challenge due to complex regeneration processes and control signals. Therefore, a single approach is difficult in obtaining satisfactory therapeutic efficacy for severe diabetic skin wounds. In this study, we adopted a composite strategy for diabetic skin wound healing. First, we fabricated a collagen-based biomimetic skin scaffold. The human basic fibroblast growth factor (bFGF) gene was electrically transduced into human umbilical cord mesenchymal stromal cells (UC-MSCs), and the stable bFGF-overexpressing UC-MSCs (bFGF-MSCs) clones were screened out. Then, an inspired collagen scaffold loaded with bFGF-MSCs was applied to treat full-thickness skin incision wounds in a streptozotocin-induced diabetic rat model. The mechanism of skin damage repair in diabetes mellitus was investigated using RNA-Seq and Western blot assays. The bioinspired collagen scaffold demonstrated good biocompatibility for skin-regeneration-associated cells such as human fibroblast (HFs) and endothelial cells (ECs). The bioinspired collagen scaffold loaded with bFGF-MSCs accelerated the diabetic full-thickness incision wound healing including cell proliferation enhancement, collagen deposition, and re-epithelialization, compared with other treatments. We also showed that the inspired skin scaffold could enhance the in vitro tube formation of ECs and the early angiogenesis process of the wound tissue in vivo . Further findings revealed enhanced angiogenic potential in ECs stimulated by bFGF-MSCs, evidenced by increased AKT phosphorylation and elevated HIF-1α and HIF-1β levels, indicating the activation of HIF-1 pathways in diabetic wound healing. Based on the superior biocompatibility and bioactivity, the novel bioinspired skin healing materials composed of the collagen scaffold and bFGF-MSCs will be promising for healing diabetic skin wounds and even other refractory tissue regenerations. The bioinspired collagen scaffold loaded with bFGF-MSCs could accelerate diabetic wound healing via neovascularization by activating HIF-1 pathways.

Craniomaxillofacial (CMF) bone injuries present a major surgical challenge and cannot heal naturally due to their large size and complex topography. We are developing a mineralized collagen scaffold that mimics extracellular matrix (ECM) features of bone. These scaffolds induce in vitro human mesenchymal stem cell (hMSC) osteogenic differentiation and in vivo bone formation without the need for exogenous osteogenic supplements. Here, we seek to enhance pro-regenerative potential via inclusion of placental-derived products in the scaffold architecture. The amnion and chorion membranes are distinct components of the placenta that each have displayed anti-inflammatory, immunomodulatory, and osteogenic properties. While potentially a powerful modification to our mineralized collagen scaffolds, the route of inclusion (matrix-immobilized or soluble) is not well understood. Here we compare the effect of introducing amnion and chorion membrane matrix versus soluble extracts derived from these membranes into the collagen scaffolds on scaffold biophysical features and resultant hMSC osteogenic activity. While inclusion of amnion and chorion matrix into the scaffold microarchitecture during fabrication does not influence their porosity, it does influence compression properties. Incorporating soluble extracts from the amnion membrane into the scaffold post-fabrication induces the highest levels of hMSC metabolic activity and equivalent mineral deposition and elution of the osteoclast inhibitor osteoprotegerin (OPG) compared to the conventional mineralized collagen scaffolds. Mineralized collagen-amnion composite scaffolds elicited enhanced early stage osteogenic gene expression (BGLAP, BMP2), increased immunomodulatory gene expression (CCL2, HGF, and MCSF) and increased angiogenic gene expression (ANGPT1, VEGFA) in hMSCs. Mineralized collagen-chorion composite scaffolds promoted immunomodulatory gene expression in hMSCs (CCL2, HGF, and IL6) while unaffecting osteogenic gene expression. Together, these findings suggest that mineralized collagen scaffolds modified using matrix derived from amnion and chorion membranes represent a promising environment conducive to craniomaxillofacial bone repair.

Endothelial cells (ECs) are central to vascular health but also interact with and regulate the immune system. Changes in endothelial state enable immune cells to migrate into the tissue to facilitate repair and fight infection. ECs modulate the function of immune cells through the expression of adhesion molecules, chemokines, major histocompatibility complex (MHC), and an array of co-stimulatory and inhibitor molecules. These interactions allow ECs to act as antigen presenting cells (APCs) and influence the outcome of immune recognition. This study elucidates how EC microenvironment, vascular cell biology, and immune response are not only connected but interdependent. More specifically, we explored how cell-substratum interactions influence EC antigen presentation and co-stimulation, and how these differences affect allorecognition in animal models of cell transplantation. Investigation of EC state was carried out using RNA sequencing while assessment of the allogeneic response includes measurements of immune cell cytotoxic ability, T cell proliferation, cytokine release, serum antibodies, and histological staining. Differences in substratum led to divergent EC phenotypes which in turn influenced immune response to transplanted cells, both due to the physical barrier of matrix-adhesion and differences in expression of surface markers. ECs grown in 2D on tissue culture plastic or in 3D on collagen scaffolds had significantly different basal levels of MHC expression, co-stimulatory and adhesion molecules. When treated with cytokines to mimic an inflammatory state, ECs did not converge to a single phenotype but rather responded differently based on their substratum. Generally, 3D ECs were more responsive to inflammatory stimuli than 2D ECs. These unique expression patterns measured in vitro also influence immune recognition in vivo. ECs grown in 2D were more likely to provoke a cytotoxic response while 3D ECs induced T cell proliferation. ECs are uniquely configured to sense not only local flow and mechanical forces but a range of markers related to systemic state, including immune function. ECs interact with immune cells with differing results depending on the environment in which the EC-lymphocyte interaction occurs. Therefore, understanding this relationship is essential to predicting and modifying the outcome of EC-immune interacts. We specifically examined the relationship between EC substratum and allorecognition. [ABSTRACT FROM AUTHOR]

In our aging society, the number of patients suffering from poorly healing bone defects increases. Bone morphogenetic proteins (BMPs) are used in the clinic to promote bone regeneration. However, poor control of BMP delivery and thus activity necessitates high doses, resulting in adverse effects and increased costs. It has been demonstrated that messenger RNA (mRNA) provides a superior alternative to protein delivery due to local uptake and prolonged expression restricted to the site of action. Here, we present the development of porous collagen scaffolds incorporating peptide-mRNA nanoparticles (NPs). Nanoparticles were generated by simply mixing aqueous solutions of the cationic cell-penetrating peptide PepFect14 (PF14) and mRNA. Peptide-mRNA complexes were uniformly distributed throughout the scaffolds, and matrices fully preserved cell attachment and viability. There was a clear dependence of protein expression on the incorporated amount of mRNA. Importantly, after lyophilization, the mRNA formulation in the collagen scaffolds retained activity also at 4 °C over two weeks. Overall, our results demonstrate that collagen scaffolds incorporating peptide-mRNA complexes hold promise as off-the-shelf functional biomaterials for applications in regenerative medicine and constitute a viable alternative to lipid-based mRNA formulations. [ABSTRACT FROM AUTHOR]

Stem cell-based therapy is considered an attractive tool to overcome the burden of liver diseases. However, efficient hepatic differentiation is still a big challenge for the research community. In this study, we explored a novel method for differentiation of bone marrow-derived mesenchymal stem cells (MSCs) into hepatic-like cells using 3D culture conditions and histone deacetylase inhibitor, sodium butyrate (NaBu). MSCs were characterized by the presence of cell surface markers via immunocytochemistry, flow cytometry, and by their potential for osteogenic, adipogenic, and chondrogenic differentiation. MSCs were treated with 1mM NaBu in 2D and 3D environments for 21 days. The hepatic differentiation was confirmed by qPCR and immunostaining. According to qPCR data, the 3D culture of NaBu-treated MSCs has shown significant upregulation of hepatic gene, CK-18 (P < 0.01), and hepatic proteins, AFP (P < 0.01) and ALB (P < 0.01). In addition, immunocytochemistry analysis showed significant increase (P < 0.05) in the acetylation of histones (H3 and H4) in NaBu-pretreated cells. It can be concluded from the study that NaBu-treated MSCs in 3D culture conditions can induce hepatic differentiation without the use of additional cytokines and growth factors. The method shown in this study represents an improved protocol for hepatic differentiation and could contribute to improvement in future cell-based therapeutics. [ABSTRACT FROM AUTHOR]

As a major component of extracellular matrixes (ECMs), collagen is an attractive biomaterial to fabricate porous scaffold for tissue engineering due to their similarity to the in vivo static microenvironment. However, the collagen-based porous scaffolds were difficult to mimic the dynamically remolded porous structure of ECM during the cell proliferation and tissue development, and always have poor mechanical property and not easy to handle. Here, natural collagen and partially denatured collagen was used to prepare the stepwise degradable hybrid bioscaffold with suitable mechanical property and dynamically remolded inner porous structure, which is desirable for the applications of tissue engineering. The collagen-based microporous scaffold was first prepared and used as physical support, then, the mechanical strength of which was reinforced by the import of the partially denatured collagen to give the hybrid bioscaffold. The fabrication conditions of the hybrid scaffolds were optimized, of which the thermal stability, mechanical property, and swelling property was explored. The stepwise enzymatic degradation process and the corresponding porous structure variation of the hybrid scaffold was confirmed by SEM and cell culture assays. [ABSTRACT FROM AUTHOR]

Articular cartilage degeneration is still an unsolved issue owing to its weak repairing capabilities, which usually result in fibrocartilage tissue formation. This fibrous tissue lacks of structural and bio‐mechanical properties, degrading over time. Currently, arthroscopic techniques and autologous transplantation are the most used clinical procedures. However, rather than restoring cartilage integrity, these methods only postpone further cartilage deterioration. Therefore, tissue engineering strategies aimed at selecting scaffolds that remarkably support the chondrogenic differentiation of human mesenchymal stem cells (hMSCs) could represent a promising solution, but they are still challenging for researchers. In this study, the influence of two different genipin (Gp) crosslinking routes on collagen (Coll)‐based scaffolds in terms of hMSCs chondrogenic differentiation and biomechanical performances was investigated. Three‐dimensional (3D) porous Coll scaffolds were fabricated by freeze‐drying techniques and were crosslinked with Gp following a "two‐step" and an in "bulk" procedure, in order to increase the physico‐mechanical stability of the structure. Chondrogenic differentiation efficacy of hMSCs and biomechanical behavior under compression forces through unconfined stress–strain tests were assessed. Coll/Gp scaffolds revealed an isotropic and highly homogeneous pore distribution along with an increase in the stiffness, also supported by the increase in the Coll denaturation temperature (Td = 57–63°C) and a significant amount of Coll and GAG deposition during the 3 weeks of chondrogenic culture. In particular, the presence of Gp in "bulk" led to a more uniform and homogenous chondral‐like matrix deposition by hMSCs if compared to the results obtained from the Gp "two‐step" functionalization procedure. [ABSTRACT FROM AUTHOR]

Wound healing-associated difficulties continue to drive biotechnological creativeness into complex grounds. The sophisticated architecture of skin wound sites and the intricate processes involved in the response to the use of regenerative devices play a critical role in successful skin regeneration approaches and their possible outcomes. Due to a plethora of complications involved in wound healing processes as well as the coordination of various cellular mechanisms, biomimetic approaches seems to be the most promising starting ground. This study evaluates the behavior of a crosslinked, porous collagen scaffold obtained by lyophilization and dehydrothermal reticulation (DHT). We address the key physio-chemical and mechanical factors, such as swelling, density and porosity, mechano-dynamic properties, SEM and TG-DSC, as well as important biological outcomes regarding scaffold biocompatibility and cellular metabolic activity, cytokine expression in inflammation, apoptosis and necrosis, as well as hemocompatibility and biodegradation. The mechanical and visco-elastic behavior are correlated, with the samples found to present similar thermal behavior and increased rigidity after DHT treatment. High biocompatibility rates were obtained, with no inflammatory stimulation and a reduction in necrotic cells. Higher percentages of cellular early apoptosis were observed. The hemocompatibility rate was under 2%, coagulation effects expressed after 4 min, and the DHT scaffold was more resistant to the biodegradation of collagenase compared with the untreated sample. [ABSTRACT FROM AUTHOR]

Endothelial cells (ECs) are central to vascular health but also interact with and regulate the immune system. Changes in endothelial state enable immune cells to migrate into the tissue to facilitate repair and fight infection. ECs modulate the function of immune cells through the expression of adhesion molecules, chemokines, major histocompatibility complex (MHC), and an array of co-stimulatory and inhibitor molecules. These interactions allow ECs to act as antigen presenting cells (APCs) and influence the outcome of immune recognition. This study elucidates how EC microenvironment, vascular cell biology, and immune response are not only connected but interdependent. More specifically, we explored how cell-substratum interactions influence EC antigen presentation and co-stimulation, and how these differences affect allorecognition in animal models of cell transplantation. Investigation of EC state was carried out using RNA sequencing while assessment of the allogeneic response includes measurements of immune cell cytotoxic ability, T cell proliferation, cytokine release, serum antibodies, and histological staining. Differences in substratum led to divergent EC phenotypes which in turn influenced immune response to transplanted cells, both due to the physical barrier of matrix-adhesion and differences in expression of surface markers. ECs grown in 2D on tissue culture plastic or in 3D on collagen scaffolds had significantly different basal levels of MHC expression, co-stimulatory and adhesion molecules. When treated with cytokines to mimic an inflammatory state, ECs did not converge to a single phenotype but rather responded differently based on their substratum. Generally, 3D ECs were more responsive to inflammatory stimuli than 2D ECs. These unique expression patterns measured in vitro also influence immune recognition in vivo. ECs grown in 2D were more likely to provoke a cytotoxic response while 3D ECs induced T cell proliferation. ECs are uniquely configured to sense not only local flow and mechanical forces but a range of markers related to systemic state, including immune function. ECs interact with immune cells with differing results depending on the environment in which the EC-lymphocyte interaction occurs. Therefore, understanding this relationship is essential to predicting and modifying the outcome of EC-immune interacts. We specifically examined the relationship between EC substratum and allorecognition.

The combination of biological scaffold and neural stem cells (NSCs) is a promising treatment strategy for spinal cord injury (SCI). But adverse niche at injures spinal cord severe hamper the transplanted NSCs to preferentially differentiate neurons. In this study, we compared the therapeutic outcomes in rat completely transected SCI model after transplantations of collagen scaffold loaded with NSCs in different forms including unicellular NSCs, neurospheres, differentiated unicellular NSCs, and differentiated neurospheres. The results showed collagen scaffold combined each form of NSCs could markedly restore the motor function of the hindlimbs by Basso-Beattie-Bresnahan(BBB) scoring to similar levels. The transcriptome analysis showed there were similar expressions of neurotrophic factors among neurospheres, differentiated unicellular NSCs, and differentiated neurospere. Next, we enriched neurotrophic factors secreted from culture medium of these three forms of NSCs respectively and combined with collagen scaffold were transplanted into SCI to further assay therapeutic effects of secreting neurotrophic factors on SCI. BBB score analysis showed that the secreted neurotrophic factors from above three forms of NSCs had similar functional recovery of SCI. Therefore, we proposed that allogeneic NSCs transplant promotes functional recovery of SCI predominantly by secreting neurotrophic factors, not by direct neuronal replacement of differentiated neurons from transplanted cells.

Introduction: This study aimed to examine the bone-forming ability of medium-cross-linked recombinant collagen peptide (mRCP) particles developedbased on human collagen type I, contains an arginyl-glycyl-aspartic acid-rich motif, fabricated as bone filling material, compared to that of the autologous bone graft. Methods: Calvarial bone defects were created in immunodeficient rats though a surgical procedure. The rats were divided into 2 groups: mRCP graft and tibia bone graft (bone graft). The bone formation potential of mRCP was evaluated by micro-computed tomography and hematoxylin-eosin staining at 1, 2, 3, and 4 weeks after surgery, and the data were analyzed and compared to those of the bone graft. Results: The axial volume-rendered images demonstrated considerable bony bridging with the mRCP graft, but there was no significant difference in the bone volume and bone mineral density between the mRCP graft and bone graft at 4 weeks. The peripheral new bone density was significantly higher than the central new bone density and the bottom side score was significantly higher than the top side score at early stage in the regenerated bone within the bone defects. Conclusion: These results indicate that mRCP has a high potential of recruiting osteogenic cells, comparable to that of autologous bone chips.

Hyaline cartilage has very limited repair capability and cannot be rebuilt predictably using conventional treatments. This study presents Autologous Chondrocyte Implantation (ACI) on two different scaffolds for the treatment of lesions in hyaline cartilage in rabbits. The first one is a commercially available scaffold (Chondro–Gide) made of collagen type I/III and the second one is a polyethersulfone (PES) synthetic membrane, manufactured by phase inversion. The revolutionary idea in the present study is the fact that we used PES membranes, which have unique features and benefits that are desirable for the 3D cultivation of chondrocytes. Sixty-four White New Zealand rabbits were used in this research. Defects penetrating into the subchondral bone were filled with or without the placement of chondrocytes on collagen or PES membranes after two weeks of culture. The expression of the gene encoding type II procollagen, a molecular marker of chondrocytes, was evaluated. Elemental analysis was performed to estimate the weight of tissue grown on the PES membrane. The reparative tissue was analyzed macroscopically and histologically after surgery at 12, 25, and 52 weeks. RT-PCR analysis of the mRNA isolated from cells detached from the polysulphonic membrane revealed the expression of type II procollagen. The elementary analysis of polysulphonic membrane slices after 2 weeks of culture with chondrocytes revealed a concentration of 0.23 mg of tissue on one part of the membrane. Macroscopic and microscopic evaluation indicated that the quality of regenerated tissue was similar after the transplantation of cells placed on polysulphonic or collagen membranes. The established method for the culture and transplantation of chondrocytes placed on polysulphonic membranes resulted in the growth of the regenerated tissue, revealing the morphology of hyaline-like cartilage to be of similar quality to collagen membranes.

The combination of scaffolds with recombinant human epidermal growth factor (rhEGF) protein can enhance defective bone healing via synergistic activation to stimulate cellular growth, differentiation, and survival. We examined the biopotentials of an rhEGF-loaded absorbable collagen scaffold (ACS) using a mouse model of calvarial defects, in which the rhEGF was produced from a plant cell suspension culture system because of several systemic advantages. Here, we showed a successful and large-scale production of plant-cell-derived rhEGF protein (p-rhEGF) by introducing an expression vector that cloned with its cDNA under the control of rice α-amylase 3D promoter into rice calli (Oryza sativa L. cv. Dongjin). Implantation with p-rhEGF (5 μg)-loaded ACSs into critical-sized calvarial defects enhanced new bone formation and the expression of osteoblast-specific markers in the defected regions greater than implantation with ACSs alone did. The potency of p-rhEGF-induced bone healing was comparable with that of Escherichia coli-derived rhEGF protein. The exogenous addition of p-rhEGF increased the proliferation of human periodontal ligament cells and augmented the induction of interleukin 8, bone morphogenetic protein 2, and vascular endothelial growth factor in the cells. Collectively, this study demonstrates the successful and convenient production of p-rhEGF, as well as its potency to enhance ACS-mediated bone regeneration by activating cellular responses that are required for wound healing.

Lack of matrix deposition is one of the main factors that complicates the healing process of wounds. The aim of this study was to test the efficacy and safety of a liquid dermal scaffold, referred to as MeshFill (MF) that can fill the complex network of tunnels and cavities which are usually found in chronic wounds and hence improve the healing process. We evaluated in vitro and in vivo properties of a novel liquid dermal scaffold in a delayed murine full‐thickness wound model. We also compared this scaffold with two commercially available granular collagen‐based products (GCBP). Liquid dermal scaffold accelerated wound closure significantly compared with no‐treated control and collagen‐based injectable composites in a delayed splinted wound model. When we compared cellular composition and count between MF, no treatment and GCBP at the histology level, it was found that MF was the most analogous and consistent with the normal anatomy of the skin. These findings were matched with the clinical outcome observation. The flowable in situ forming scaffold is liquid at cold temperature and gels after application to the wound site. Therefore, it would conform to the topography of the wound when liquid and provides adequate tensile strength when solidified. This patient‐ready gelling dermal scaffold also contains the nutritional ingredients and therefore supports cell growth. Applying an injectable liquid scaffold that can fill wound gaps and generate a matrix to promote keratinocytes and fibroblasts migration, can result in improvement of the healing process of complex wounds. [ABSTRACT FROM AUTHOR]

Spinal cord injury (SCI) often results in an inhibitory environment at the injury site. In our previous studies, transplantation of a scaffold combined with stem cells was proven to induce neural regeneration in animal models of complete SCI. Based on these preclinical studies, collagen scaffolds loaded with the patients' own bone marrow mononuclear cells or human umbilical cord mesenchymal stem cells were transplanted into SCI patients. Fifteen patients with acute complete SCI and 51 patients with chronic complete SCI were enrolled and followed up for 2 to 5 years. No serious adverse events related to functional scaffold transplantation were observed. Among the patients with acute SCI, five patients achieved expansion of their sensory positions and six patients recovered sensation in the bowel or bladder. Additionally, four patients regained voluntary walking ability accompanied by reconnection of neural signal transduction. Among patients with chronic SCI, 16 patients achieved expansion of their sensation level and 30 patients experienced enhanced reflexive defecation sensation or increased skin sweating below the injury site. Nearly half of the patients with chronic cervical SCI developed enhanced finger activity. These long-term follow-up results suggest that functional scaffold transplantation may represent a feasible treatment for patients with complete SCI. [ABSTRACT FROM AUTHOR]

Menstrual blood mesenchymal stem cell (MBMSC) is a potential cell source for effective therapy for intrauterine adhesion (IUA). Collagen scaffold (CS) loaded with mesenchymal stem cells promotes endometrial regeneration in IUA model animals. However, role of combination of MBMSCs and CS in IUA therapy remains elusive. In particular, transplantation of MBMSCs over a long period of time requires more in-depth research. Here in this study, transplantation of human MBMSCs loaded on CS was applied for therapy for a long term rat IUA model. A rat IUA model characterized by lower number of endometrial glands and increased fibrosis was established. At 90 days after transplantation of the human MBMSC-loaded CS, expression of HuNu, a human protein, was identified in the uteri of the transplanted IUA model rats. The transplantation increased the number of endometrial glands and decreased the fibrotic areas significantly. Moreover, transplantation of the human MBMSC-loaded CS decreased the Collagen I and increased the CK 18 significantly. Immunoblotting assay results further proved the downregulation of Collagen I and the upregulation of CK 18. Together, endometrium regeneration promoted by human MBMSC-loaded CS was demonstrated in a long term rat model of IUA, shedding a new light on the role of human MBMSCs in the therapy for IUA • A rat IUA model is characterized by the lower number of glands and increased fibrosis. • Transplantation of the human MBMSC-loaded CS increased the gland numbers. • Transplantation of the human MBMSC-loaded CS decreased the fibrotic areas. • Transplantation of the human MBMSC-loaded CS decreased the Collagen I. • Transplantation of the human MBMSC-loaded CS increased the CK 18. [ABSTRACT FROM AUTHOR]

This study reports the formulation of mupirocin-loaded chitosan microspheres embedded in Piper betle extract containing collagen scaffold as combinational drug delivery for improved wound healing. Selection of chitosan type (molecular weight and degree of deacetylation) was carried out based on their antibacterial efficacy. The low molecular weight chitosan was selected owing to the highest antibacterial action against gram-positive as well as gram-negative bacteria. Low molecular weight chitosan-microspheres showed spherical shape with largely smooth surface morphology, 11.81% of mupirocin loading, and its controlled release profile. The XRD, DSC thermograms, and FT-IR spectral analysis revealed the mupirocin loaded in molecularly dispersed or in amorphous form, and having no chemical interactions with the chitosan matrix, respectively. The in vivo study indicates potential effect of the mupirocin, Piper betle, and chitosan in the collagen scaffold in the wound healing efficiency with approximately 90% wound healing observed at the end of 15 days of study for combinational drug-loaded chitosan microspheres-collagen scaffold-treated group. The histopathology examination further revealed tissue lined by stratified squamous epithelium, collagen deposition, fibroblastic proliferation, and absence of inflammation indicating relatively efficient wound healing once treated with combinational drug-loaded chitosan microspheres containing scaffold. [ABSTRACT FROM AUTHOR]