Siew L. Toh

Anterior cruciate ligament regeneration using mesenchymal stem cells and silk scaffold in large animal model

Although in vivo studies in small animal model show the ligament regeneration by implanting mesenchymal stem cells (MSCs) and silk scaffold, large animal studies are still needed to evaluate the silk scaffold before starting a clinical trial. The aim of this study is to regenerate anterior cruciate ligament (ACL) in pig model. The micro-porous silk mesh was fabricated by incorporating silk sponges into knitted silk mesh with lyophilization. Then the scaffold was prepared by rolling the micro-porous silk mesh around a braided silk cord to produce a tightly wound shaft. In vitro study indicated that MSCs proliferated profusely on scaffold and differentiated into fibroblast-like cells by expressing collagen I, collagen III and tenascin-C genes in mRNA level. Then the MSCs-seeded scaffold was implanted in pig model to regenerate ACL. At 24 weeks postoperatively, the MSCs in regenerated ligament exhibited fibroblast morphology. The key ligament-specific extracellular matrix components were produced prominently and indirect ligament-bone insertion with three zones (bone, Sharpeys fibers and ligament) was observed. Although there was remarkable scaffold degradation, the maximum tensile load of regenerated ligament could be maintained after 24 weeks of implantation. In conclusion, the results imply that silk-based material has great potentials for clinical applications.

In vivo study of anterior cruciate ligament regeneration using mesenchymal stem cells and silk scaffold

Although most in vitro studies indicate that silk is a suitable biomaterial for ligament tissue engineering, in vivo studies of implanted silk scaffolds for ligament reconstruction are still lacking. The objective of this study is to investigate anterior cruciate ligament (ACL) regeneration using mesenchymal stem cells (MSCs) and silk scaffold. The scaffold was fabricated by incorporating microporous silk sponges into knitted silk mesh, which mimicked the structures of ligament extracellular matrix (ECM). In vitro culture demonstrated that MSCs on scaffolds proliferated vigorously and produced abundant collagen. The transcription levels of ligament-specific genes also increased with time. Then MSCs/scaffold was implanted to regenerate ACL in vivo. After 24 weeks, histology observation showed that MSCs were distributed throughout the regenerated ligament and exhibited fibroblast morphology. The key ligament ECM components including collagen I, collagen III, and tenascin-C were produced prominently. Furthermore, direct ligament-bone insertion with typical four zones (bone, mineralized fibrocartilage, fibrocartilage, ligament) was reconstructed, which resembled the native structure of ACL-bone insertion. The tensile strength of regenerated ligament also met the mechanical requirements. Moreover, its histological grading score was significantly higher than that of control. In conclusion, the results imply that silk scaffold has great potentials in future clinical applications.

Biomechanical study on axillary crutches during single-leg swing-through gait

This paper describes a kinetic and kinematic study on axillary crutches during one-leg swing-through gait. The primary objective is to evaluate the interplay of forces at the crutch and body interfaces and to relate them in the understanding of problems associated with the use of axillary crutches. Ten normal adult male subjects with simulated left leg impairment participated in the study. For data acquisition, the VICON kinematic system, a Kistler force plate and an instrumented crutch (with force transducers at the two upper struts close to the axillary bar and one near the crutch tip) were used. Results showed that the peak ground reaction force on the weight-bearing leg during lower limb stance increased by 21.6 percent bodyweight. The peak reaction force transmitted to the arm during crutch stancc was 44.4 percent bodyweight. These increased loadings could be detrimental to patients with unsound weight-bearing leg and upper extremities respectively. When the crutches were used incorrectly, 34 percent bodyweight was carried by the underarm. This could cause undue pressure over the neurovascular structures at the axillary region.

Novel Silk Scaffolds for Ligament Tissue Engineering Applications

Scaffold technology is integral in advancing tissue engineering and one of the tissues of interest here is the tendon/ligament. Advancement in the tissue engineering of tendon/ligament has become very much a materials engineering problem than ever, with the selection of appropriate biomaterial and scaffold architecture. Such is the key to successful tendon/ligament tissue regeneration construct. Popular materials used in recent years include various poly (l-lactic) biomaterials and collagen. However, shortcomings of these materials, in terms of poor mechanical strength or short degradation period, are yet overcome. Bombyx mori silk, though used in biomedical sutures for decades due to its excellent mechanical properties, has been overlooked for applications in ligament tissue engineering, only until recently. This is largely due to previous misconceptions in its biocompatibility and biodegradability characteristics. This paper describes the use of a silk-based scaffold with knitted architecture and investigates its strengths as compared to previous PLGA-based knitted scaffolds. An electrospun nanofiber surface on knitted microfiber architecture is adopted and it is found to have better composite-material integrity, in vitro degradation resistance, and encourages cell adhesion and proliferation.

Characterization of Electrospun Substrates for Ligament Regeneration using Bone Marrow Stromal Cells

Challenges persist in the tissue engineering and regeneration of ligaments. Of the various aspects contributing to the success of ligament tissue engineering (such as the scaffold, cell source and stimulatory biochemical and mechanical cues), architecture and material involved in scaffold design remain as a significant area of study. Essentially, the scaffold for ligament regeneration, especially the cell-seed substrate, should be viable for cell attachment, promotes nutrient and waste transfer, be mechanically viable, and stimulates initial cell proliferation and ECM production. In this study, electrospun substrates using the silk fibroin (SF) and PLGA material, with different electrospun fiber arrangements (aligned (AL) and random (RD)) were compared for their ability to promote MSC attachment and subsequent differentiation down the ligament fibroblast cell lineage. The rationale for such characterizations lies in the hypothesis that SF, being a natural protein, provides significantly more favorable surface chemistry for cell attachment and differentiation than synthetic polymers such as PLGA. On the other hand, the aligned electrospun fiber arrangement will mimic the native ligament ECM environment, to guide MSCs down the fibroblast lineage. Electrospun PLGA and SF substrates were fabricated by first dissolving the respective material in HFIP (7–10% w/v). Following which, aligned PLGA and SF substrates were collected from a rotational electrospin setup, while the random types were collected from a grounded plate. These four groups of substrates (SF-AL, SF-RD, PLGA-AL, PLGA-RD) were then characterized in terms of cell adhesion, proliferation, viability and function via post-seed cell counting, AlamarBlueTM assay, Fluorescence Microscopy, Sircol® Collagen Assay and SEM. Results from this characterization show that cells remained viable when cultured on these substrates, with the AL types being mechanically stronger and promoted increased proliferation and collagen production as compared to the RD types. SF substrates promoted cell attachment and differentiation, as inferred from the increased collagen production.

Ligament-to-bone Interface Tissue Regeneration Using a Functionalized Biphasic Silk Fibroin Scaffold

The emergence of the tissue engineering approach has shown to be a game changer in ligament reconstruction, making it possible to create multi-phasic scaffold structures for multi-specific tissue regeneration. To regenerate the bone-ligament-bone tissue, mimicking hard to soft tissue transition, we propose the use of a functionalized biphasic silk scaffold. Hydroxyapatite nanoparticles (nHA) and bone morphogenetic protein 2 (BMP2) were loaded in the ends of Bombyx mori silk fibroin (SF) scaffold system to enhance enthesis regeneration and bone tunnel healing, while the central onethird supported ligament regeneration. Two groups of biphasic scaffolds, distinguished by the different ends’ additive, were fabricated: nHA only (Ctrl) and n-HA/BMP2 (Exp). A series of bench work, small animal study and large animal preclinical trial was performed using MSC-seeded constructs for the reconstruction of excised ACL. The bioactivity of BMP2 was ascertained and shown to be eluting with an initial burst, followed by a lowered sustained release. Osteogenic genes were upregulated in both groups compared to pure SF. By 24 weeks in vivo, the ACL was regenerated and bone tunnel narrowing was observed with histological evidences indicating new bone and enthesis regeneration in Exp. Better graft to bone integration was observed in Exp compared to Ctrl. From this study, it was demonstrated that the BMP2 eluting biphasic silk scaffold is promising as an advanced tissue engineering treatment modality for complete bone-ligament-bone reconstruction.

Establishing a Coculture System for Ligament-Bone Interface Tissue Engineering

Ligament-bone interface (enthesis) is a complex structure which comprises of ligament, fibrocartilage and bone. The fibrocartilage transformation adds significant insertional strength to the interface and makes it highly resistant to avulsion forces. Many ACL grafts cannot generate native interfacial region, leading to their failure. Co-culture has proved to be an effective way to generate new tissues in tissue engineering. Studies have found important signaling molecules in transduction pathway of chondrogenesis to be transmitted via gap junctions. We hypothesized that stem cells cocultured between ligament and bone cells would enable transmission of chondrogenic factors from bone/ligament cells to bone marrow stem cells (BMSCs) via gap junctions, resulting in their differentiation into fibrocartilage. To test this hypothesis, we studied to establish effective co-culture system. In this study, two set of co-culture (BMSCs and ligament cells; BMSCs and bone cells) were established. Confocal microscopy showed efficient dye transfer from bone/ligament cells into BMSCs. This was further confirmed and quantified by FACS, which showed a gradual temporal increase in the percentage of BMSCs acquiring Calcein. RT-PCR analysis showed that the bone cells-BMSC and the ligament cells-BMSC co-culture systems expressed higher amounts of collagen type-2, as compared to the various monocultures. The results proved the establishment of effective co-culture. The findings provide important information for the development of a more promising ligamentfibrocartilagebone graft.