Low-level laser therapy (780 nm) combined with collagen sponge scaffold promotes repair of rat cranial critical-size defects and increases TGF-ß, 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 780 nm; total energy density 120 J/cm2 ; power 50 mW) alone (G2); and CSS associated with LLLT treatment (G3). After surgery, animals in each group were euthanized at 21 days and 30 days (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-ß), 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-ß, and the most intense and diffuse OCN immunofluorescent labelling at 30 days 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.

The Rotary Cell Culture System increases NTRK3 expression and promotes neuronal differentiation and migratory ability of neural stem cells cultured on collagen sponge.

BACKGROUND: Recently, neural stem cell (NSC) therapy has shown promise for the treatment of many neurological diseases. Enhancing the quality of implanted cells and improving therapeutic efficacy are currently research hotspots. It has been reported that collagen sponge material provided sufficient room for cell growth in all directions and promoted the absorption of nutrients and removal of wastes. And also, the Rotary Cell Culture System (RCCS), which mimics the microgravity environment, can be used to culture cells for tissue engineering. MATERIALS AND METHODS: We performed the mRNA and miRNA sequencing to elucidate the regulatory mechanism of NSCs cultured on the collagen sponge in the RCCS system. The luciferase assay and Western blot revealed a direct regulatory role between let-7i-5p and neurotrophic receptor tyrosine kinase 3 (NTRK3; also called TrkC). And then, the neural differentiation markers Tuj1 and Map2 were detected by immunofluorescence staining. In the meantime, the migratory ability of NSCs was detected both in vitro and in spinal cord injury animals. RESULTS: In this study, we demonstrated that the expression of NTRK3 was elevated in NSCs cultured on collagen sponge in the RCCS system. Furthermore, increased NTRK3 expression was regulated by the downregulation of let-7i-5p. Compared to traditionally cultured NSCs, the NSCs cultured on collagen sponge in the RCCS system exhibited better neuronal differentiation and migratory ability, especially in the presence of NT-3. CONCLUSIONS: As the biological properties and quality of transplanted cells are critical for therapeutic success, the RCCS system combined with the collagen sponge culture system shows promise for applications in clinical practice in the future.

Epithelial differentiation of human adipose-derived stem cells (hASCs) undergoing three-dimensional (3D) cultivation with collagen sponge scaffold (CSS) via an indirect co-culture strategy.

BACKGROUND: Three-dimensional (3D) cultivation with biomaterials was proposed to facilitate stem cell epithelial differentiation for wound healing. However, whether human adipose-derived stem cells (hASCs) on collagen sponge scaffold (CSS) better differentiate to keratinocytes remains unclear. METHODS: 3D cultivation with CSS on hASC epidermal differentiation co-cultured with HaCaT cells at air-liquid interface (ALI) was compared with two-dimensional (2D) form and cultivation without "co-culture" or "ALI." Cellular morphology, cell adhesion, and growth condition were evaluated, followed by the protein and gene expression of keratin 14 (K14, keratinocyte specific marker). RESULTS: Typical cobblestone morphology of keratinocytes was remarkably observed in co-cultured hASCs at ALI, but those seeded on the CSS exhibited more keratinocyte-like cells under an invert microscope and scanning electron microscope. Desired cell adhesion and proliferation were confirmed in 3D differentiation groups by rhodamine-labeled phalloidin staining, consistent with H&E staining. Compared with those cultured in 2D culture system or without "ALI," immunofluorescence staining and gene expression analysis revealed hASCs co-cultured over CSS expressed K14 at higher levels at day 15. CONCLUSIONS: CSS is positive to promote epithelial differentiation of hASCs, which will foster a deeper understanding of artificial dermis in skin wound healing and regeneration.

Comparison of Regenerative Effects of Transplanting Three-Dimensional Longitudinal Scaffold Loaded-Human Mesenchymal Stem Cells and Human Neural Stem Cells on Spinal Cord Completely Transected Rats.

Stem cell-based therapy has been considered as a potential treatment to restore spinal cord injury (SCI) through reconstructing neural networks and providing a favorable microenvironment for neuronal survival, differentiation, and axonal outgrowth. Biomaterial scaffolds can promote cell attachment and survival, neuronal differentiation, and axonal outgrowth; therefore, they were used to combine with stem cells for implantation in SCI treatment. In addition, a longitudinal scaffold can guide regenerated axons with orientated growth and axial extension. Both human umbilical cord-derived mesenchymal stem cells (hMSCs) and human fetal spinal cord-derived neural stem cells (hNSCs) have been applied in clinical trials worldwide. To our knowledge, a parallel comparison of the therapeutic effects of hMSC and hNSC implantations has not been conducted. Hence, in this study, we grafted hMSCs or hNSCs seeded on longitudinal collagen sponge scaffolds into rats with completely transected SCI to examine differences in SCI repair. Both hMSCs and hNSCs had equivalent effects on reducing glial scar formation around the lesion gap. More neuronal class III ß-tubulin-positive neurons and neurofilament-positive nerve fibers were found in the lesion cavity after hNSC implantation. In addition, hNSCs had better capabilities to improve motor function, attenuate inflammation, and promote cell survival than hMSCs. These encouraging results provide a clinical basis for future stem cell-based SCI therapies.

Knitted Silk-Collagen Scaffold Incorporated with Ligament Stem/Progenitor Cells Sheet for Anterior Cruciate Ligament Reconstruction and Osteoarthritis Prevention.

Current surgical management of anterior cruciate ligament (ACL) rupture still remains an intractable challenge in ACL regeneration due to the weak self-healing capability of ACL. Inadequate cell numbers and vascularization within the articular cavity contribute mainly to the poor prognosis. This time, we fabricated a new tissue engineering scaffold by adding ligament stem/progenitor cell (LSPC) sheets to our previous knitted silk-collagen sponge scaffold, which overcame these limitations by providing sufficient numbers of seed cells and a natural extracellular matrix to facilitate regeneration. LSPCs display excellent proliferation and multilineage differentiation capacity. Upon ectopic implantation, the knitted silk-collagen sponge scaffold incorporated with an LSPC sheet exhibited less immune cells but more fibroblast-like cells, deposited ECM and neovascularization, and better tissue ingrowth. In a rabbit model, we excised the ACL and performed a reconstructive surgery with our scaffold. Increased expression of ligament-specific genes and better collagen fibril formation could be observed after orthotopic transplantation. After 6 months, the LSPC sheet group showed better results on ligament regeneration and ligament-bone healing. Furthermore, no obvious cartilage and meniscus degeneration were observed at 6 months postoperation. In conclusion, these results indicated that the new tissue engineering scaffold can promote ACL regeneration and slow down the progression of osteoarthritis, thus suggesting its high clinical potential as an ideal graft in ACL reconstruction.

Construction of tendon replacement tissue based on collagen sponge and mesenchymal stem cells by coupled mechano-chemical induction and evaluation of its tendon repair abilities.

Tissue engineering is an ideal therapeutic strategy for the development of functional tendon replacement tissue for tendon repair in the clinic. Currently, the synergistic roles of mechano-chemical factors and the mechanisms involved in tendon repair and regeneration are not fully understood. In this study, we developed a three-dimensional (3D) culture system based on a silicone chamber and collagen sponge scaffold that can deliver cyclic mechanical stretch and biochemical stimulation to bone marrow-derived mesenchymal stem cells (BMSCs) seeded on the scaffold. We found that the combined stimulation of cyclic stretch and transforming growth factor beta 1 (TGF-ß1) treatment not only increased cell viability but also synergistically promoted the differentiation of BMSCs into tenocytes in a 3D culture environment. Meanwhile, the combined stimulation increased the Young's modulus of the BMSC-collagen sponge constructs by reducing the porosity of the scaffold compared to the non-treated constructs. Furthermore, a rat Achilles tendon in situ repair experiment showed that enhanced tendon regeneration was achieved using the BMSC-collagen sponge construct combined with cyclic stretch and TGF-ß1, as confirmed by Achilles functional index (AFI) measurement, morphological observation, histological analysis, and mechanical testing. These results suggest that this approach could offer a practical benefit in tendon healing and future tendon tissue engineering. STATEMENT OF SIGNIFICANCE: This study aims to disclose the crucial roles of the coupled induction by mechano-chemical stimulation in tendon tissue engineering and clarifies their collaborative control mechanisms. We developed a three-dimensional (3D) culture system based on a silicone chamber and collagen sponge scaffold that could deliver cyclic mechanical stretch and biochemical stimulation to bone marrow-derived mesenchymal stem cells (BMSCs). We found that the combined stimulation of cyclic stretch and transforming growth factor beta 1 (TGF-ß1) could result in an improvement of tissue-engineered construct for enhancing tendon healing. These results suggest that this approach could offer a practical benefit in tendon healing and future tendon tissue engineering.

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.