In vitro generation of whole osteochondral constructs using rabbit bone marrow stromal cells, employing a two-chambered co-culture well design.

The regeneration of whole osteochondral constructs with a physiological structure has been a significant issue, both clinically and academically. In this study, we present a method using rabbit bone marrow stromal cells (BMSCs) cultured on a silk-RADA peptide scaffold in a specially designed two-chambered co-culture well for the generation of multilayered osteochondral constructs in vitro. This specially designed two-chambered well can simultaneously provide osteogenic and chondrogenic stimulation to cells located in different regions of the scaffold. We demonstrated that this co-culture approach could successfully provide specific chemical stimulation to BMSCs located on different layers within a single scaffold, resulting in the formation of multilayered osteochondral constructs containing cartilage-like and subchondral bone-like tissue, as well as the intermediate osteochondral interface. The cells in the intermediate region were found to be hypertrophic chondrocytes, embedded in a calcified extracellular matrix containing glycosaminoglycans and collagen types I, II and X. In conclusion, this study provides a single-step approach that highlights the feasibility of rabbit BMSCs as a single-cell source for multilayered osteochondral construct generation in vitro.

Enhanced osteoinductivity and osteoconductivity through hydroxyapatite coating of silk-based tissue-engineered ligament scaffold.

Hybrid silk scaffolds combining knitted silk fibers and silk sponge have been recently developed for use as ligament-alone grafts. Incorporating an osteoinductive phase into the ends of a ligament scaffold may potentially generate an integrated "bone-ligament-bone" graft and improve graft osteointegration with host bone. To explore the possible application of hydroxyapatite (HA) coating in the fabrication of osteoinductive ends of silk-based scaffold, HA was coated on the hybrid silk scaffold and the effects to the bone-related cells were evaluated. HA could be coated in a uniform and controlled manner on the silk sponge, using an alternate soaking technology, with the amount deposited being dependent on the number of soaking cycles. HA coating also progressively reduced the hydrophobicity of silk surface (decreasing water contact angle from 87° to 42-76°, after 1-3 soaking cycles), making the HA-coated silk scaffold less favorable for initial cell attachments; but the attached cells showed viability and sustained proliferation on the HA-coated scaffold. As demonstrated by real-time polymerase chain reaction and alkaline phosphatase assay, the osteoinductivity of HA-coated silk scaffolds resulted in the osteogenic differentiation of bone marrow mesenchymal stem cells, and the osteoconductivity of HA-coated silk scaffolds supported osteoblasts growth and maintained the properties of mature osteoblasts. These properties of HA-coating demonstrated its possible application in fabricating osteoinductive ends of the silk-based ligament graft to potentially enhance graft-to-host bone integration.

In vitro ligament-bone interface regeneration using a trilineage coculture system on a hybrid silk scaffold.

The ligament-bone interface is a complex structure that comprises ligament, fibrocartilage, and bone. We hypothesize that mesenchymal stem cells cocultured in between ligament and bone cells, on a hybrid silk scaffold with sections suitable for each cell type, would differentiate into fibrocartilage. The section of scaffold for osteoblast seeding was coated with hydroxyapatite. A trilineage coculture system (osteoblasts-BMSCs-fibroblasts) on a hybrid silk scaffold was established. RT-PCR results and immunohistochemistry results demonstrated that BMSCs cocultured between fibroblasts and osteoblasts had differentiated into the fibrocartilaginous lineage. The morphological change was also observed by SEM observation. A gradual transition from the uncalcified to the calcified region was formed in the cocultured BMSCs from the region that directly interacted with fibroblasts to the region that directly interacted with osteoblasts. The role of transforming growth factor ß3 (TGF-ß3) in this trilineage coculture model was also investigated by supplementing the coculture system with 10 ng/mL TGF-ß3. The TGF-treated group showed similar results of fibrocartilaginous differentiation of BMSCs with coculture group without TGF-ß3 supplement. However, no calcium deposition was found in the cocultured BMSCs in the TGF-treated group. This may indicate TGF-ß3 delayed the mineralization process of chondrocytes.

A hybrid silk/RADA-based fibrous scaffold with triple hierarchy for ligament regeneration.

While silk-based microfibrous scaffolds possess excellent mechanical properties and have been used for ligament tissue-engineering applications, the microenvironment in these scaffolds is not biomimetic. We hypothesized that coating a hybrid silk scaffold with an extracellular matrix (ECM)-like network of self-assembling peptide nanofibers would provide a biomimetic three-dimensional nanofibrous microenvironment and enhance ligament tissue regeneration after bone marrow-derived mesenchymal stem cell (BMSC)-seeding. A novel scaffold possessing a triple structural hierarchy comprising macrofibrous knitted silk fibers, a silk microsponge, and a peptide nanofiber mesh was developed by coating self-assembled RADA16 peptide nanofibers on a silk microfiber-reinforced-sponge scaffold. Compared with the uncoated control, RADA-coated scaffolds showed enhanced BMSC proliferation, metabolism, and fibroblastic differentiation during the 3 weeks of culture. BMSC-seeded RADA-coated scaffolds showed an increasing temporal expression of key fibroblastic ECM proteins (collagen type I and III, tenascin-C), with a significantly higher tenascin-C expression compared with the controls. BMSC-seeded RADA-coated scaffolds also showed a temporal increase in total collagen and glycosaminoglycan production (the amount produced being higher than in control scaffolds) during 3 weeks of culture, and possessed 7% higher maximum tensile load compared with the BMSC-seeded control scaffolds. The results indicate that the BMSC-seeded RADA-coated hybrid silk scaffold system has the potential for use in ligament tissue-engineering applications.

Anterior cruciate ligament failure and cartilage damage during knee joint compression: a preliminary study based on the porcine model.

BACKGROUND: Anterior cruciate ligament (ACL) injury incurred from high-impact activities leads to an increased risk of osteoarthritis. HYPOTHESIS: Impact forces that cause ACL failure can also inflict cartilage damage, whereby its extent and distribution may be influenced by the ligament failure mechanism. STUDY DESIGN: Descriptive laboratory study. METHODS: Six porcine knee specimens were mounted to a material testing system at 70 degrees of flexion. During compression, rotational and translational data of the specimens were recorded with a motion-capture system. Compression was successively repeated with increasing actuator displacement until a significant drop in compressive force response was observed; ligament failure was assessed by dissection. Osteocartilage explants were extracted from the meniscus-covered sites (anterior, exterior, and posterior) and exposed (interior) sites on both tibial compartments. The explants were sectioned, stained, and histologically scored using the modified Mankin grading system. RESULTS: Five of the 6 specimens incurred ACL failure. On failure, a significant compressive force drop (1812.5-2659.3 N) was observed together with considerable posterior femoral translation; 2 specimens underwent external rotation, while 2 had internal rotation and 1 had no substantial rotation. Generally, the meniscus-covered sites displayed significant surface fraying and occasional deep clefts; the exposed site did not present substantial surface irregularities but indicated more tidemark disruption. Higher Mankin scores observed at certain sites illustrated a localized presence of contact and shear forces, which may be caused by pivoting and sliding of the femoral condyles during rotation. CONCLUSION: The porcine model can be a tenable preliminary option for assessing the role of the human ACL during joint compression. Impact loads that result in ligament failure can potentially inflict considerable cartilage damage; the damage profile may be affected by the type of failure mechanism. CLINICAL RELEVANCE: Cartilage injury arising at the time of ACL injury may lead to an accelerated risk of joint degeneration.