James Cho Hong Goh

A bFGF-releasing silk/PLGA-based biohybrid scaffold for ligament/tendon tissue engineering using mesenchymal progenitor cells

An ideal scaffold that provides a combination of suitable mechanical properties along with biological signals is required for successful ligament/tendon regeneration in mesenchymal stem cell-based tissue engineering strategies. Among the various fibre-based scaffolds that have been used, hybrid fibrous scaffolds comprising both microfibres and nanofibres have been recently shown to be particularly promising. This study developed a biohybrid fibrous scaffold system by coating bioactive bFGF-releasing ultrafine PLGA fibres over mechanically robust slowly-degrading degummed knitted microfibrous silk scaffolds. On the ECM-like biomimetic architecture of ultrafine fibres, sustained release of bFGF mimicked the ECM in function, initially stimulating mesenchymal progenitor cell (MPC) proliferation, and subsequently, their tenogeneic differentiation. The biohybrid scaffold system not only facilitated MPC attachment and promoted cell proliferation, with cells growing both on ultrafine PLGA fibres and silk microfibres, but also stimulated tenogeneic differentiation of seeded MPCs. Upregulated gene expression of ligament/tendon-specific ECM proteins and increased collagen production likely contributed to enhancing mechanical properties of the constructs, generating a ligament/tendon analogue that has the potential to be used to repair injured ligaments/tendons.

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.

Selection of Cell Source for Ligament Tissue Engineering

Use of appropriate types of cells could potentially improve the functionality and structure of tissue engineered constructs, but little is known about the optimal cell source for ligament tissue engineering. The object of this study was to determine the optimal cell source for anterior cruciate ligament (ACL) tissue engineering. Fibroblasts isolated from anterior cruciate ligament, medial collateral ligament (MCL), as well as bone marrow mesenchymal stem cells (MSC) were compared using the following parameters: proliferation rate, collagen excretion, expression of collagen type I, II, and III, as well as α-smooth muscle actin. Green fluorescent protein (GFP) transfected MSCs were used to trace their fate in the knee joints. MSC, ACL, and MCL fibroblasts were all highly stained with antibodies for collagen types I and III and α-smooth muscle actin while negatively stained with collagen type II. Proliferation rate and collagen excretion of MSCs were higher than ACL and MCL fibroblasts (p < 0.05), and MSCs could survive for at least 6 weeks in knee joints. In summary, MSC is potentially a better cell source than ACL and MCL fibroblasts for anterior cruciate ligament tissue engineering.

PLGA nanofiber-coated silk microfibrous scaffold for connective tissue engineering.

A modified degumming technique, involving boiling in 0.25% Na2CO3 with addition of 1% sodium dodecyl sulphate and intermittent ultrasonic agitation, was developed for knitted silk scaffolds. Sericin was efficiently removed, while mechanical and structural properties of native silk fibroin were preserved. Biocompatible and mechanically robust hybrid nano-microscaffolds were fabricated by coating these degummed silk scaffolds with an intervening adhesive layer of silk solution followed by electrospun poly-lactic-co-glycolic acid (PLGA) nanofibers. Cell proliferation on the hybrid silk scaffolds was improved by seeding cells on both surfaces of the flat scaffolds. Rolling up and continued culture of the cell-seeded hybrid scaffolds yielded cylindrical constructs that permitted cell proliferation, extracellular matrix deposition, and generated ligament/tendon graft analogs. Although PLGA-based hybrid scaffolds have earlier been proposed for dense connective tissue engineering, rapid biodegradation of PLGA was a drawback. In contrast, the underlying strong and slowly-degrading microfibrous silk scaffold used in this study ensured that the hybrid scaffold maintained adequate mechanical properties for longer periods, which is vital for continued support to the injured ligament/tendon throughout its healing period.

The efficacy of bone marrow stromal cell-seeded knitted PLGA fiber scaffold for Achilles tendon repair.

Tendons are frequently targets of injury in sports and aging trauma. Significant dysfunction and disability can result from suboptimal healing of tendon injuries. It is generally thought that tendons have some self-healing ability, but in cases where tendons are missing or the wound sites are too large to allow for reapposition of the ends, tendon replacement is necessary.1 Various bioabsorbable materials have been used for Achilles regeneration;2 however, no published data that we are aware of has addressed the use of D,L-lactide-co-glycolide (PLGA, 10:90) fibers or knitted structure to deliver bone marrow stromal cells for tendon repair. This study was conducted to evaluate the effect of marrow-stromal-cell (bMSC)-seeded knitted PLGA scaffold for Achilles tendon repair in a rabbit model.

Viability of allogeneic bone marrow stromal cells following local delivery into patella tendon in rabbit model.

Bone marrow stromal cells are potentially useful for tendon repair and regeneration. To provide lasting benefits, the seeded cells must survive implantation at local tendon sites. Our objective was to determine the in vivo fate of allogeneic bone marrow stromal cells (bMSCs) at different time points after implantation into patella tendon defects (i.e., at 2, 3, 5, and 8 weeks). The protocol involved the labeling of bMSCs with green fluorescent protein (GFP) or carboxyfluorescein diacetate (CFDA) before implantation. A window defect (5 × 5 mm) was created at the central portion of rabbit patella tendon and subsequently treated with GFP- or CFDA-marked bMSCs. The marked bMSCs were loaded into the window defect with fibrin glue. Upon sacrifice of the rabbits at the different time points, the implant site of the patellar tendon was immediately retrieved and the viability of the labeled cells was assessed under confocal microscopy. The results showed that the seeded bMSCs remained viable within the tendon wound site for at least 8 weeks after implantation. The cell morphology was changed from a round shape at 2 weeks to a spindle shape at 5 weeks after implantation. This study demonstrated that the bMSCs remained viable for prolonged periods after implantation and therefore have the potential to influence the formation and remodeling of neotendon tissue after tendon repair.

The effects of bone marrow-derived mesenchymal stem cells and fascia wrap application to anterior cruciate ligament tissue engineering.

After an anterior cruciate ligament (ACL) injury, surgical reconstructions are necessary in most cases, either with autografts, allografts, or artificial ligaments. Potential tissue-engineered ligaments would circumvent the disadvantages apparent in these methods. While seeding of mesenchymal stem cells (MSCs) and fascia wrap could potentially improve tissue regeneration and mechanical properties, their exact roles were evaluated in the current study. Knitted biodegradable scaffolds of poly-L-lactic acid (PLLA) and poly-glycolic-lactic acid (PGLA) yarns were used to reconstruct ACL in 48 rabbits. These were divided into four equal groups: only knitted scaffolds were used in group I; knitted scaffolds and mesenchymal stem cells were used in group II; knitted scaffolds, MSCs, and fascia lata were used in group III; knitted scaffolds and fascia lata were used in group IV. Carboxyfluorescein diacetate (CFDA)-labeled MSCs were used to trace the fate of seeded cells in groups II and III. Histology, Western blot analysis, and mechanical properties of reconstructed ACL were analyzed after 20 weeks. Fibroblast ingrowths were seen in all four groups while CFDA-labeled MSCs could be found after 8 weeks of implantation in groups II and III. Both the amount of collagen type I and collagen type III in groups III and IV were significantly higher than in group II, which was much higher than in group I. Both maximal tensile loads and stiffness of the reconstructed ACLs in groups I, II, III, and IV were significantly lower than normal controls after 20 weeks of implantation. It is concluded that MSCs could promote synthesis of collagen type I and collagen type III in tissue-engineered ligaments, while fascia wraps have stronger effects. Both MSC seeding and fascia wrap could not enhance ultimate tensile load and stiffness.

Characterization of anterior cruciate ligament cells and bone marrow stromal cells on various biodegradable polymeric films

In this study, the adhesion, proliferation and morphology of rabbit anterior cruciate ligament (ACL) cells and bone marrow stromal cells (bMSCs) on synthetic biodegradable polymeric films were investigated. Tissue culture polystyrene (TCP) was used as control. Seven biodegradable polymers were used; they are as follows: poly(e-caprolactone) (PCL), poly(DL-lactide) (D-PLA), poly(L-lactide) (L-PLA), PLA/PCL (50:50), PLA/PCL (75:25), high molecular weight (HMW) poly(DL-lactide-co-glycolide (PLGA50:50) and HMW PLGA75:25. Polymeric film substrates were manufactured using solvent spin-casting technique. After 8 h of cell culture, a high percentage of ACL cells was found attached to PLGA50:50 (38.6 ± 8.4%) and TCP (39.3 ± 6.1%) as compared to the other six polymeric films (p ≤ 0.001). As for bMSCs, 76.4 ± 10%, 76.3 ± 16% and 76.1 ± 19% of seeded bMSCs were adhered to TCP, PLGA50:50 and PLGA75:25, respectively. These were significantly more than those of the other five polymeric films (p<0.001). At Day 5, bMSCs were found to proliferate faster on TCP (by 7 ± 0.8-fold of initial cell seeding number), D-PLA (by 5.6 ± 1.6-fold), PLGA50:50 (by 9.3 ± 1.3-fold) and PLGA75:25 (by 5.8 ± 1.3-fold) than on PCL, PLLA and PCL/PLA (50:50, 25:75) (p<0.001). ACL cells had a greater fold expansion on TCP (by 3.5 ± 0.2-fold), PLGA50:50 (by 3.1 ± 0.4-fold) and PLGA75:25 (by 3.9 ± 0.4-fold) than on the other five polymer substrates (p <0.001). From these results, HMW PLGA (50:50, 75:25) was shown more likely to allow bMSCs and ACL cells to attach and proliferate, and bMSCs attached and proliferated faster than ACL cells.

Bio-Electrospraying: A Potentially Safe Technique for Delivering Progenitor Cells

Bio‐electrospraying is fast becoming an attractive tool for in situ cell delivery into scaffolds for tissue engineering applications, with several cell types been successfully electrosprayed. Bone marrow derived mesenchymal progenitor/stem cells (BMSC), which are an important cell source for tissue engineering, have not been explored in detail and the effect of electrospraying on their “stemness” is not known. This study therefore investigates the effects of electrospraying on BMSC viability, proliferation, and multilineage differentiation potential. Electrospraying a BMSC suspension at flow rate of 6u2009mL/h and voltages of 7.5–15u2009kV could successfully generate a continuous, stable and linearly directed electrospray of cells. Morphological observation, trypan blue tests and alamar blue based metabolic assays revealed about 88% of these electrosprayed cells were viable, and proliferated at rates similar to native BMSCs. However, at higher voltages, electrospraying became unstable and reduced cell viability, possibly due to electrical or thermal damage to the cells. BMSCs electrosprayed at 7.5u2009kV also retained their multipotency and could be successfully differentiated into adipogenic, chondrogenic, and osteogenic lineages, demonstrating similar morphology and gene expression levels as induced native BMSCs. These results indicate that bio‐electrospraying could be safely used as a progenitor/stem cell delivery technique for tissue engineering and regenerative medicine applications. Biotechnol. Bioeng. 2010;106: 690–698.