Collagen Sponge Scaffold Research Articles

Regulation of Extracellular Matrix Synthesis by Shell Extracts from the Marine Bivalve Pecten maximus in Human Articular Chondrocytes- Application for Cartilage Engineering.

The shells of the bivalve mollusks are organo-mineral structures predominantly composed of calcium carbonate, but also of a minor organic matrix, a mixture of proteins, glycoproteins, and polysaccharides. These proteins are involved in mineral deposition and, more generally, in the spatial organization of the shell crystallites in well-defined microstructures. In this work, we extracted different organic shell extracts (acid-soluble matrix, acid-insoluble matrix, water-soluble matrix, guanidine HCl/EDTA-extracted matrix, referred as ASM, AIM, WSM, and EDTAM, respectively) from the shell of the scallop Pecten maximus and studied their biological activities on human articular chondrocytes (HACs). We found that these extracts differentially modulate the biological activities of HACs, depending on the type of extraction and the concentration used. Furthermore, we showed that, unlike ASM and AIM, WSM promotes maintenance of the chondrocyte phenotype in monolayer culture. WSM increased the expression of chondrocyte-specific markers (aggrecan and type II collagen), without enhancing that of the main chondrocyte dedifferentiation marker (type I collagen). We also demonstrated that WSM could favor redifferentiation of chondrocyte in collagen sponge scaffold in hypoxia. Thus, this study suggests that the organic matrix of Pecten maximus, particularly WSM, may contain interesting molecules with chondrogenic effects. Our research emphasizes the potential use of WSM of Pecten maximus for cell therapy of cartilage.

Mechanical barriers and transforming growth factor beta inhibitor on epidural fibrosis in a rabbit laminectomy model

BackgroundTGF-β has been described as a mediator of fibrosis and scarring. Several studies achieved reduction in experimental scarring through the inhibition of TGF-β. Fibroblasts have been defined as the cell population originating fibrosis, blocking fibroblast invasion may impair epidural fibrosis appearance. For this purpose, biocompatible materials used as mechanical barriers and a TGF-β inhibitor peptide were evaluated in the reduction of epidural fibrosis.MethodsA L6 laminectomy was performed in 40 New Zealand white rabbits. Divided into four groups, each rabbit was assigned to receive either collagen sponge scaffold (CS group), gelatin-based gel (GCP group), P144® (iTGFβ group), or left untreated (control group). Four weeks after surgery, cell density, collagen content, and new bone formation of the scar area were determined by histomorphometry. Two experienced pathologists scored dura mater adhesion, scar density, and inflammatory infiltrate in a blinded manner.ResultsIn all groups, laminectomy site was filled with fibrous tissue and the dura mater presented adhesions. Only GCP group presented a significant reduction in collagen content and scar density.ConclusionGCP treatment reduces epidural fibrosis although did not prevent dura mater adhesion completely.

Characterization and evaluation of graphene oxide scaffold for periodontal wound healing of class II furcation defects in dog.

IntroductionThe 3-dimensional scaffold plays a key role in volume and quality of repair tissue in periodontal tissue engineering therapy. We fabricated a novel 3D collagen scaffold containing carbon-based 2-dimensional layered material, named graphene oxide (GO). The aim of this study was to characterize and assess GO scaffold for periodontal tissue healing of class II furcation defects in dog.Materials and methodsGO scaffolds were prepared by coating the surface of a 3D collagen sponge scaffold with GO dispersion. Scaffolds were characterized using cytotoxicity and tissue reactivity tests. In addition, GO scaffold was implanted into dog class II furcation defects and periodontal healing was investigated at 4 weeks postsurgery.ResultsGO scaffold exhibited low cytotoxicity and enhanced cellular ingrowth behavior and rat bone forming ability. In addition, GO scaffold stimulated healing of dog class II furcation defects. Periodontal attachment formation, including alveolar bone, periodontal ligament-like tissue, and cementum-like tissue, was significantly increased by GO scaffold implantation, compared with untreated scaffold.ConclusionThe results suggest that GO scaffold is biocompatible and possesses excellent bone and periodontal tissue formation ability. Therefore, GO scaffold would be beneficial for periodontal tissue engineering therapy.

Tenogenic differentiation of mesenchymal stem cells improves healing of linea alba incision

OBJECTIVE: The aim of this study is to investigate the curative effects of mesenchymal stem cells' (MSCs') tenogenic differentiation on Linea alba incision healing. MATERIALS AND METHODS: Autologous MSCs were isolated from rat bone marrow and cultured and induced by 10 ng/mL of bone morphogenetic protein-12 (BMP-12) for 48 h. Expression of scleraxis (SCX), collagen I, and collagen III was examined at 48 h, 5 days, and 7 days to investigate the tenogenic differentiation. Fifty Sprague-Dawley rats were randomly divided into five groups: tenogenically differentiated (group E) or native mesenchymal stem cells (group D) seeded onto collagen sponge scaffolds or only sponge scaffolds (group C) were transplanted into the linea alba incision; rats that underwent operation without implantation of anything served as the sham group (group B), and rats that did not undergo operation were used as the control group (group A). Histological analysis was performed to explore the curative effects. RESULTS: The expression of SCX increases continually even in the absence of BMP-12 for 5 days (P CONCLUSIONS: In animal experiments, MSCs' tenogenic differentiation induced by BMP-12 can dramatically enhance linea alba incision healing.

Collagen Sponge Scaffold for Tissue-Engineered Skeletal Muscle

Collagen Sponge Scaffold for Tissue-Engineered Skeletal Muscle

Evaluation of the bony repair in rat cranial defect using near infrared reflectance spectroscopy and discriminant analysis.

We set out to determine whether near infrared reflectance spectroscopy (NIRS) combined with principal component analysis-linear discriminant analysis (LDA) or, variable selection techniques employing successive projection algorithm or genetic algorithm (GA) could evaluate the bone repair in cranial critical-size (5 mm) defect after stimulation with collagen sponge scaffold and/or infrared low-level laser therapy directly on the local. Forty-five Winstar rats were divided into nine groups of five each, namely: group H - healthy, n = 5 (without treatment and without cranial critical-size defect), (GI positive control - n = 5, 21 days or n = 5, 30 days) without treatment and with cranial critical-size defect; (GII-n = 5, 21 days or n = 5, 30 days) cranial critical-size defect filled with collagen sponge scaffold; (GIII-n = 5, 21 days or n = 5, 30 days) cranial critical-size defect submitted to low-level laser therapy; (GIV-n = 5, 21 days or n = 5, 30 days) cranial critical-size defect submitted to combined collagen sponge scaffold + low-level laser therapy treatment. In relation to the histological analysis, the collagen sponge scaffold + low-level laser therapy treatment group (GIV) 30 days showed the best result with the presence of secondary bone, immature bone (osteoid) and newly formed connective tissue (periosteum). GA-LDA model also successfully classified control class of the others classes. Thus, the results provided by the good-quality classification model revealed the feasibility of NIRS for application to evaluation of the wound healing in rat cranial defect, thanks to the short analysis time of a few seconds and nondestructive advantages of NIRS as an alternative approach for bone repair purposes. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:1160-1168, 2017.

Low-level laser therapy (780nm) combined with collagen sponge scaffold promotes repair of rat cranial critical-size defects and increases TGF-β, FGF-2, OPG/RANK and osteocalcin expression.

The aim of this study was to evaluate the effect of collagen sponge scaffold (CSS) implantation associated with low-level laser therapy (LLLT) on repairing bone defects. A single 5-mm cranial defect was surgically created in forty Wistar rats, which then received one of the following four interventions (n=10 per group): no treatment (G0); bone defect implanted with collagen sponge scaffold (CSS) alone (G1); defect treated with low-level laser therapy (LLLT) (wavelength 780nm; total energy density 120J/cm2 ; power 50mW) alone (G2); and CSS associated with LLLT treatment (G3). After surgery, animals in each group were euthanized at 21days and 30days (n=5 per euthanasia time group). Bone formation was monitored by X-ray imaging analysis. Biopsies were collected and processed for histological analysis and immunohistochemical evaluation of transforming growth factor-beta (TGF-β), fibroblast growth factor-2 (FGF-2), osteoprotegerin (OPG) and receptor activator of nuclear factor ƙ (RANK). Osteocalcin (OCN) was detected by immunofluorescence analysis. Compared to the G0 group, defects in the 30-day G3 group exhibited increased bone formation, both by increase in radiopaque areas (P<0.01) and by histomorphometric analysis (P<0.001). The histopathological analysis showed a decreased number of inflammatory cells (P<0.001). The combined CCS+LLLT (G3) treatment also resulted in the most intense immunostaining for OPG, RANK, FGF-2 and TGF-β, and the most intense and diffuse OCN immunofluorescent labelling at 30days postsurgery (G3 vs. G0 group, P<0.05). Therefore, the use of CCS associated with LLLT could offer a synergistic advantage in improving the healing of bone fractures.

Efficacy of 50 Hz electromagnetic fields on human epidermal stem cell transplantation seeded in collagen sponge scaffolds for wound healing in a murine model

To explore the possible efficacy of electromagnetic fields (EMF) for skin tissue engineering, effects of EMF exposure on epidermal stem cells (ESC) seeded in collagen sponge scaffolds for wound healing in a murine model were investigated. The wound models of a full-thickness defect established with 36 7 ∼ 8-week-old nude mice were randomly divided into three groups: a control group, an ESC-only group, and an ESC with EMF exposure group (frequency of 50 Hz, magnetic induction of 5 mT, 60 min per day for 20 days). ESC were separated from human foreskin and cultured in vitro, and then transplanted with collagen sponge scaffolds as a delivery vehicle to wounds of the ESC-only group, and ESC with EMF exposure group was exposed to EMF after ESC transplantation. Effects of EMF on morphological changes and expression of β1 integrin in regenerated skins were observed. Wound healing rates and healing times were collected to evaluate the efficacy of repairment. Results showed that human ESC were successfully transplanted to nude mice, which facilitated the formation of intact skin on nude mice. In contrast to other groups, the wound healing of ESC with EMF exposure group was the fastest (P < 0.05), the structure of regenerated skins was more mature, and it contained more continuity in the number of viable cell layers and rich hair follicles' structure. These results suggest that the use of 50 Hz EMF as a non-invasive treatment can accelerate wound healing of ESC transplantation, and restore structural integrity of regenerated skin. Bioelectromagnetics. 38:204-212,2017. © 2017 Wiley Periodicals, Inc.

Heparin cross-linked collagen sponge scaffolds improve functional regeneration of rat tracheal epithelium.

Tracheal epithelial cells maintain airway homeostasis by mediating mucociliary clearance. Following tracheal reconstruction, timely epithelial regeneration is required to prevent respiratory compromise and infectious diseases. To achieve rapid tracheal epithelial regeneration, a heparin cross-linked collagen sponge containing fibroblast growth factor-2 (FGF-2) was prepared as a graft for tracheal reconstruction. The heparin cross-linked sponge exhibited a high FGF-2 retaining capacity, and tracheal epithelial and mesenchymal cells cultured in this sponge containing FGF-2 showed high proliferative capacities. Subsequently, heparin-free collagen sponge scaffolds (C/F scaffold) and collagen sponge scaffolds cross-linked with 10μg/ml heparin retained FGF-2 (C/H10/F scaffold), and were transplanted into rats with tracheal defects. Invasion of both epithelial and non-epithelial cells was greater in rats treated with the C/H10/F scaffold at 1week post-transplantation than in rats treated with the C/F scaffold. Moreover, at 2weeks after transplantation, improved cilia formation was observed in the C/H10/F scaffold group, with higher motility and more potent posterior-anterior flow generation than in the C/F scaffold group. These results suggest that heparin improves functional regeneration of tracheal epithelium. Copyright © 2017 John Wiley & Sons, Ltd.

Physical and mechanical properties of cross-linked type I collagen scaffolds derived from bovine, porcine, and ovine tendons.

Collagen scaffolds are often utilized in tissue engineering applications where their performance depends on physical and mechanical properties. This study investigated the effects of collagen source (bovine, porcine, and ovine tendon) on properties of collagen sponge scaffolds cross-linked with 1-ethyl-3-(3-dimethyl aminopropyl) carbodiimide (EDC)/N-hydroxysuccinimide (NHS). Scaffolds were tested for tensile and compressive properties, stability (resistance to enzymatic degradation), pore size, and swelling ratio. No significant differences in tensile modulus were observed, but ovine scaffolds had significantly greater ultimate strain, stress, and toughness relative to bovine and porcine scaffolds. No significant differences in compressive properties, pore size, or swelling ratio were observed as a function of collagen source. Ovine scaffolds were more resistant to collagenase degradation compared to bovine samples, which were more resistant than porcine scaffolds. In comparison to bovine scaffolds, ovine scaffolds performed equivalently or superiorly in all evaluations, and porcine scaffolds were equivalent in all properties except enzymatic stability. These results suggest that collagen sponges derived from bovine, porcine, and ovine tendon have similar physical and mechanical properties, and are all potentially suitable materials for various tissue engineering applications. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 2685-2692, 2016.

Graphene oxide scaffold accelerates cellular proliferative response and alveolar bone healing of tooth extraction socket.

Graphene oxide (GO) consisting of a carbon monolayer has been widely investigated for tissue engineering platforms because of its unique properties. For this study, we fabricated a GO-applied scaffold and assessed the cellular and tissue behaviors in the scaffold. A preclinical test was conducted to ascertain whether the GO scaffold promoted bone induction in dog tooth extraction sockets. For this study, GO scaffolds were prepared by coating the surface of a collagen sponge scaffold with 0.1 and 1 µg/mL GO dispersion. Scaffolds were characterized using scanning electron microscopy (SEM), physical testing, cell seeding, and rat subcutaneous implant testing. Then a GO scaffold was implanted into a dog tooth extraction socket. Histological observations were made at 2 weeks postsurgery. SEM observations show that GO attached to the surface of collagen scaffold struts. The GO scaffold exhibited an interconnected structure resembling that of control subjects. GO application improved the physical strength, enzyme resistance, and adsorption of calcium and proteins. Cytocompatibility tests showed that GO application significantly increased osteoblastic MC3T3-E1 cell proliferation. In addition, an assessment of rat subcutaneous tissue response revealed that implantation of 1 µg/mL GO scaffold stimulated cellular ingrowth behavior, suggesting that the GO scaffold exhibited good biocompatibility. The tissue ingrowth area and DNA contents of 1 µg/mL GO scaffold were, respectively, approximately 2.5-fold and 1.4-fold greater than those of the control. Particularly, the infiltration of ED2-positive (M2) macrophages and blood vessels were prominent in the GO scaffold. Dog bone-formation tests showed that 1 µg/mL GO scaffold implantation enhanced bone formation. New bone formation following GO scaffold implantation was enhanced fivefold compared to that in control subjects. These results suggest that GO was biocompatible and had high bone-formation capability for the scaffold. The GO scaffold is expected to be beneficial for bone tissue engineering therapy.

Tissue-engineered skeletal muscle with collagen sponge scaffold having contractile properties

Event Abstract Back to Event Tissue-engineered skeletal muscle with collagen sponge scaffold having contractile properties Shunya Takagi1, Tomohiro Nakamura1 and Toshia Fujisato1 1 Osaka Institute of Technology, Department of Biomedical Engineering, Japan Introduction: A tissue-engineered skeletal muscle is a promising strategy for the reconstruction of skeletal muscle loss caused by tumour ablations and by accidental injury. In the process of skeletal muscle regeneration, myoblasts fuse each other to form multinucleated myotubes, and then the myotubes mature to myofiber in vivo. The primary function of skeletal muscle is to generate force. We have already prepared the tissue-engineered skeletal muscle of three-dimensional structure having diameter of about 0.5 mm and length of 12 mm by the collagen gel cell culture. However, it is still a big challenge to construct a matured muscle tissue in vitro having applicable mass to tissue transplantation. As tissue constructs increase in scale, their size is limited by poor nutrient supply. The current major issue is a lack of angio architecture in the tissue-engineered constructs. In this study, a collagen scaffold having uniaxial porous structure was reported for the large-scaled and has function native-like contractile properties by electrical stimulations for tissue-engineered skeletal muscle. Materials and Methods : The polytetrafluorethylene tube was filled with Type I collagen gel solution. It was placed vertically and frozen by gradual immersion into liquid nitrogen from its bottom to top. The frozen collagen gel solution was then dried in a vacuum oven. The obtained sponge scaffold was subjected to the SEM observation. The C2C12 established myoblasts were seeded into the collagen sponge scaffold. The construct was cultured for 2 days in DMEM containing fetal bovine serum and then the medium was changed to DMEM containing horse serum to enhance differentiation of the cells to the myotubes. The contractile force of tissue-engineered skeletal muscle caused by electric stimulation was measured after cultures. Then the construct was observed histologically by HE staining. Results and Discussion: The obtained collagen sponge was shown to have unidirectional porous structure. This structure may be created by ice crystals formed vertically in the collagen solution by gradual freezing by liquid nitrogen. The C2C12 cells were dispersed throughout the structure of the collagen sponge. However tissue-engineered skeletal muscle observed C2C12 cells gradually becomes one line. Tissue-engineered skeletal muscle the reaction was in response to the twitch and tetanus stimulation applied. Fig.1 Histological evaluation of collagen sponge scaffold. (A) The SEM image of collagen sponge scaffold. Bar = 200 µm. (B) The HE staining of cultured tissue-engineered skeletal muscle. Bar = 100 µm. Conclusions: From these results, the collagen sponge having unidirectional porous structure may be useful for giving large mass tissue engineered constructs. Collagen sponge has native-like contractile properties by electrical stimulations. Keywords: Cell Differentiation, Tissue Engineering, in vivo Conference: 10th World Biomaterials Congress, Montréal, Canada, 17 May - 22 May, 2016. Presentation Type: Poster Topic: Biomaterials in constructing tissue substitutes Citation: Takagi S, Nakamura T and Fujisato T (2016). Tissue-engineered skeletal muscle with collagen sponge scaffold having contractile properties. Front. Bioeng. Biotechnol. Conference Abstract: 10th World Biomaterials Congress. doi: 10.3389/conf.FBIOE.2016.01.00651 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 27 Mar 2016; Published Online: 30 Mar 2016. Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Shunya Takagi Tomohiro Nakamura Toshia Fujisato Google Shunya Takagi Tomohiro Nakamura Toshia Fujisato Google Scholar Shunya Takagi Tomohiro Nakamura Toshia Fujisato PubMed Shunya Takagi Tomohiro Nakamura Toshia Fujisato Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

Effect of Structural Differences in Collagen Sponge Scaffolds on Tracheal Epithelium Regeneration.

We developed an in situ regeneration-inducible artificial trachea composed of a porcine collagen sponge and polypropylene framework and used it for tracheal reconstruction. In the present study, collagen sponges with different structures were prepared from various concentrations of collagen solutions, and their effect on the regeneration of tracheal epithelium was examined. Collagen sponges were prepared from type I and III collagen solutions. The structures of the sponges were analyzed using scanning electron microscopy (SEM). Artificial tracheae, which were formed using the collagen sponges with different structures, were implanted into rabbits, and regeneration of the tracheal epithelium on the artificial tracheae was evaluated by SEM analysis and histological examination. The SEM analysis showed that collagen sponges prepared from 0.5% and 1.0% collagen solutions had a porous structure. However, the sponges prepared from a 1.5% collagen solution had a nonporous structure. After implantation of artificial tracheae prepared from 0.5% and 1.0% collagen solutions, their luminal surfaces were mostly covered with epithelium within 14 days. However, epithelial reorganization occurred later on artificial tracheae prepared from the 1.5% collagen solution. Collagen sponges with a porous structure are suitable for regeneration of the tracheal epithelium in our artificial trachea.

Bone formation by human umbilical cord perivascular cells.

We investigated the possibility of employing human umbilical perivascular cells (HUCPVCs) within the context of finding an alternative source of mesenchymal stromal cells (MSC) for bone tissue engineering. Since it has previously been reported that conditioned medium (CM) from osteogenic bone marrow (BM) MSCs can potentiate osteogenic differentiation in a secondary cell population, we also employed BM-MSCs to generate CM to stimulate osteogenesis in the HUCPVCs. The BM-MSCs were a commercially available immortalized human cell line. In vitro assays showed negligible levels of osteogenic gene expression in HUCPVCs compared to BM-MSC, but alkaline phosphatase was detected when HUCPVC were cultured in osteogenic medium in the presence of CM from BM-MSC. An in vivo assay employing a rat calvarial osteotomy defect, together with a collagen sponge scaffold, showed that HUCPVCs provided statistically significant bony repair compared to controls. BM-MSC loaded scaffolds were not statistically different from either controls or HUCPVCs. The addition of BM-MSC CM to HUCPVCs also produced no statistically significant difference to the bone formed by HUCPVCs alone. Our results demonstrate that the in vitro assays employed did not predict in vivo outcomes, and that the BM-MSC cell line employed, or CM from such cells, provided no osteogenic advantage over the use of HUCPVCs alone.

A 3D nanofibrous hydrogel and collagen sponge scaffold promotes locomotor functional recovery, spinal repair, and neuronal regeneration after complete transection of the spinal cord in adult rats

Central nervous system neurons in adult mammals display limited regeneration after injury, and functional recovery is poor following complete transection (>4 mm gap) of a rat spinal cord. A novel combination scaffold composed of 3D nanofibrous hydrogel PuraMatrix and a honeycomb collagen sponge was used to promote spinal repair and locomotor functional recovery following complete transection of the spinal cord in rats. We transplanted this scaffold into 5 mm spinal cord gaps and assessed spinal repair and functional recovery using the Basso, Beattie, and Bresnahan (BBB) locomotor scale. The BBB score of the scaffold-transplanted group was significantly higher than that of the PBS-injected control group from 24 d to 4 months after the operation (P < 0.001–0.01), reaching 6.0 ± 0.75 (mean ± SEM) in the transplant and 0.70 ± 0.46 in the control groups. Neuronal regeneration and spinal repair were examined histologically using Pan Neuronal Marker, glial fibrillary acidic protein, growth-associated protein 43, and DAPI. The scaffolds were well integrated into the spinal cords, filling the 5 mm gaps with higher numbers of regenerated and migrated neurons, astrocytes, and other cells than in the control group. Mature and immature neurons and astrocytes in the scaffolds became colocalized and aligned longitudinally over >2 mm, suggesting their differentiation, maturation, and function. The spinal cord NF200 content of the transplant group, analyzed by western blot, was more than twice that of the control group, supporting the histological results. Transplantation of this novel scaffold promoted functional recovery, spinal repair, and neuronal regeneration.

Frequency-Dependence of Mechanically Stimulated Osteoblastic Calcification in Tissue-Engineered Bone In Vitro.

The effect of mechanical stimulation on osteogenesis remains controversial, especially with respect to the loading frequency that maximizes osteogenesis. Mechanical stimulation at an optimized frequency may be beneficial for the bone tissue regeneration to promote osteoblastic calcification. The objective of this study was to investigate the frequency-dependent effect of mechanical loading on osteoblastic calcification in the tissue-engineered bones in vitro. Tissue-engineered bones were constructed by seeding rat osteoblasts into a type I collagen sponge scaffold at a cell density of 1600 or 24,000cells/mm(3). Sinusoidal compressive deformation at the peak of 0.2% was applied to the tissue-engineered bones at 0.2, 2, 10, 20, 40, and 60Hz for 3min/day for 14 consecutive days. Optically-monitored calcium content started to increase on days 5-7 and reached the highest value at 2Hz on day 14; however, no increase was observed at 0.2Hz and in the control. Ash content measured after the mechanical stimulation also showed the highest at 2Hz despite the differences in cell seeding density. It was concluded that mechanical stimulation at 2Hz showed the highest promotional effect for osteogenesis in vitro among the frequencies selected in this study.

HBMP-2 and hTGF-β1 expressed in implanted BMSCs synergistically promote the repairing of segmental bone defects

ObjectiveTo evaluate the effects of co-expressing hBMP-2 and hTGF-β31 in BMSCs (bone marrow-derived mesenchymal stem cells) on the repairing process of radial segmental defects in rats. MethodsBMSCs were infected with a high titer recombinant adenovirus carrying hTGF-βl and/or hBMP-2 genes. Expression of exogenous genes in BMSCs was confirmed by RT-PCR and ELISA assays. In vitro effects of exogenous genes were assessed by MTT and ALP activity tests. A left radial defect model was created using 120 SD rats. Genetically modified or unmodified BMSCs were implanted with collagen sponge scaffolds into the 5-mm radial defect. The bone repair process was systematically monitored and evaluated by X-ray examinations, gross anatomic examinations, histological analyses, and biomechanical tests. ResultsExpression of hBMP-2 and hTGF-β1 showed synergistic effects on promoting BMSC proliferation and enhancing ALP activity in vitro. Bone repair assays showed that hBMP-2 and hTGF-β1 promoted the production of chondrocytes and osteoblasts. Implanted BMSCs transfected with both hBMP-2 and hTGF-β1 led to the best bone repair outcome.

Long-term effects of knitted silk–collagen sponge scaffold on anterior cruciate ligament reconstruction and osteoarthritis prevention

Anterior cruciate ligament (ACL) is difficult to heal after injury due to the dynamic fluid environment of joint. Previously, we have achieved satisfactory regeneration of subcutaneous tendon/ligament with knitted silk–collagen sponge scaffold due to its specific “internal-space-preservation” property. This study aims to investigate the long-term effects of knitted silk–collagen sponge scaffold on ACL regeneration and osteoarthritis prevention. The knitted silk–collagen sponge scaffold was fabricated and implanted into a rabbit ACL injury model. The knitted silk–collagen sponge scaffold was found to enhance migration and adhesion of spindle-shaped cells into the scaffold at 2 months post-surgery. After 6 months, ACL treated with the knitted silk–collagen sponge scaffold exhibited increased expression of ligament genes and better microstructural morphology. After 18 months, the knitted silk–collagen sponge scaffold-treated group had more mature ligament structure and direct ligament-to-bone healing. Implanted knitted silk–collagen sponge scaffolds degraded much more slowly compared to subcutaneous implantation. Furthermore, the knitted silk–collagen sponge scaffold effectively protected joint surface cartilage and preserved joint space for up to 18 months post-surgery. These findings thus demonstrated that the knitted silk–collagen sponge scaffold can regenerate functional ACL and prevent osteoarthritis in the long-term, suggesting its clinical use as a functional bioscaffold for ACL reconstruction.

Characterization of scaffold carriers for BMP9-transduced osteoblastic progenitor cells in bone regeneration

Successful bone tissue engineering at least requires sufficient osteoblast progenitors, efficient osteoinductive factors, and biocompatible scaffolding materials. We have demonstrated that BMP9 is one of the most potent factors in inducing osteogenic differentiation of mesenchymal progenitors. To facilitate the potential use of cell-based BMP9 gene therapy for bone regeneration, we characterize the in vivo osteoconductive activities and bone regeneration potential of three clinically used scaffold materials, type I collagen sponge, hydroxyapatite-tricalcium phosphate (HA-TCP), and demineralized bone matrix (DBM), using BMP9-expressing C2C12 osteoblastic progenitor cells. We find that recombinant adenovirus-mediated BMP9 expression effectively induces osteogenic differentiation in C2C12 cells. Although direct subcutaneous injection of BMP9-transduced C2C12 cells forms ectopic bony masses, subcutaneous implantation of BMP9-expressing C2C12 cells with collagen sponge or HA-TCP scaffold yields the most robust and mature cancellous bone formation, whereas the DBM carrier group forms no or minimal bone masses. Our results suggest that collagen sponge and HA-TCP scaffold carriers may provide more cell-friendly environment to support the survival, propagation, and ultimately differentiation of BMP9-expressing progenitor cells. This line of investigation should provide important experimental evidence for further preclinical studies in BMP9-mediated cell-based approach to bone tissue engineering.

Improvements of osteoblast adhesion, proliferation, and differentiation in vitro via fibrin network formation in collagen sponge scaffold

Collagen sponges (COL-S) are used as scaffolds to support osteoblasts and stimulate bone repair because of their flexibility, biocompatibility, and biodegradability. In this study, we added fibrin networks to COL-S scaffolds by using a fibrinogen (FNG) cross-linking reaction and evaluated the proliferation, differentiation, and adhesion of MG-63 cells on these scaffolds. The fibrin network that formed in COL-S with various concentrations of FNG was characterized with regard to morphology, porosity, and water-uptake ability. Successful fibrin network formation was observed by scanning electron microscopy (SEM). As the FNG concentration increased, network formation increased, but porosity and water-uptake ability were slightly reduced at high FNG concentrations. An MTS assay, DNA content assay, live/dead fluorescence assay, and SEM imaging showed that MG-63 cells attached and spread on COL-S and COL-S/FNG scaffolds, particularly on scaffolds modified using FNG. In addition, alkaline phosphatase (ALP) activity was significantly increased in cells cultured on scaffolds modified using 10, 40, and 80 mg/mL FNG. Thus, the addition of a fibrin network could increase the biocompatibility of COL-S for bone regeneration.