Young Jick Kim

Injectable extracellular matrix hydrogel developed using porcine articular cartilage

This work was first development of a delivery system capable of maintaining a sustained release of protein drugs at specific sites by using potentially biocompatible porcine articular cartilage. The prepared porcine articular cartilage powder (PCP) was easily soluble in phosphate-buffered saline. The PCP suspension easily entrapped bovine serum albumin-fluorescein isothiocyanate (BSA-FITC) in pharmaceutical formulations at room temperature. The aggregation of PCP and BSA-FITC was confirmed by dynamic light scattering. When the BSA-FITC-loaded PCP suspension was subcutaneously injected into rats, it gelled and formed an interconnecting three-dimensional PCP structure that allowed BSA to penetrate through it. The amount of BSA-FITC released from the PCP hydrogel was determined in rat plasma and monitored by real-time in vivo molecular imaging. The data indicated sustained release of BSA-FITC for 20 days in vivo. In addition, the PCP hydrogel induced a slight inflammatory response. In conclusion, we showed that the PCP hydrogel could serve as a minimally invasive therapeutics depot.

Effect of different bone marrow stimulation techniques (BSTs) on MSCs mobilization

The therapeutic effect of bone marrow stimulation techniques (BSTs) is mainly attributed to the role of mesenchymal stem cells (MSCs) from the bone marrow. However, no studies have directly shown the amount of MSCs drained by BSTs. This study hypothesized that differences in the opening of subchondral bone affect the number of MSCs drained from the bone marrow. We purposed that as the exposed area and hole size of BSTs vary, the number of MSCs drained out was measured. Three groups of different BSTs were designed that have variations in the sizes of total exposed area and individual holes. Three different BSTs using a curette, 1.5‐ and 0.8‐mm awls were carried out on the full‐thickness femoral cartilage defect of young rabbits. After BST, the number of MSCs in the blood clot was measured by CFU‐Fs assay. As the size of the total exposed area increased, so did the number of MSCs obtained. The number of MSCs drained from bone marrow may vary depending on different BSTs and this could affect therapeutic efficacy of cartilage defect. As current microfracture (MF) method cannot drain the most MSCs clinically, more improved surgery technique is needed.

Implantation of bone marrow-derived buffy coat can supplement bone marrow stimulation for articular cartilage repair

OBJECTIVE Bone marrow stimulation (BMS) has been regarded as a first line procedure for repair of articular cartilage. However, repaired cartilage from BMS is known to be unlike that of hyaline cartilage and its inner endurance is not guaranteed. The reason presumably came from a shortage of cartilage-forming cells in blood clots derived by BMS. In order to increase repairable cellularity, the feasibility of autologous bone marrow-derived buffy coat transplantation in repair of large full-thickness cartilage defects was investigated in this study. METHODS Rabbits were divided into four groups: the defect remained untreated as a negative control; performance of BMS only (BMS group); BMS followed by supplementation of autologous bone marrow buffy coat (Buffy coat group); transplantation of autologous osteochondral transplantation (AOTS) as a positive control. RESULTS Repair of cartilage defects in the Buffy coat group in a rabbit model was more effective than BMS alone and similar to AOTS. Gross findings, histological analysis, histological scoring, immunohistochemistry, and chemical assay demonstrated that supplementation of autologous bone marrow buffy coat after BMS arthroplasty effectively repaired cartilage defects in a rabbit model, and was more effective than BMS arthroplasty alone. CONCLUSION Supplementation of autologous bone marrow-derived buffy coat in cases of BMS could be a useful clinical protocol for cartilage repair.

Histomorphochemical comparison of microfracture as a first‐line and a salvage procedure: Is microfracture still a viable option for knee cartilage repair in a salvage situation?

Microfracture is considered as the first‐line procedure for knee cartilage repair, but the results of microfracture seem less predictable and rather controversial in a salvage situation. Thus, the purpose of the study was to histomorphochemically compare microfracture as a salvage procedure with microfracture as a first‐line procedure in a rabbit model. We hypothesized that microfracture in a salvage situation would result in histomorphochemically inferior cartilage repair compared to microfracture as a first‐line procedure, and the inferiority would be attributed to less migration of reparable marrow cells to the defect due to destruction of microarchitecture of the subchondral bone. Thirty‐six New Zealand white rabbits were divided into three groups: (i) untreated full‐thickness chondral defect, (ii) single microfracture treatment (first microfracture group), and (iii) repeated microfracture in 8 weeks after the first procedure (second microfracture group). In each group, rabbits were sacrificed at the end of 8 weeks, and osteochondral specimens at the repair sites were obtained for histomorphochemical analysis. Results showed that microfracture as a salvage procedure resulted in overall inferior cartilage repair histomorphochemically compared with microfracture as a first‐line procedure, which correlated with deteriorative changes in the quality of underlying subchondral bone rather than intrinsic incapability to recruit the reparative cells in the defect area. In conclusion, although a comparable number of reparable cells and a mechanically weakened subchondral bone are anticipated, more study is necessary to clearly determine when a microfracture should be performed in a situation.

In vivo degradation profile of porcine cartilage-derived extracellular matrix powder scaffolds using a non-invasive fluorescence imaging method

Abstract We present a non-invasive fluorescence method for imaging of scaffold degradation in vivo by quantifying the degradation of porcine cartilage-derived extracellular matrix powder (PCP).Three-dimensional porous scaffolds should be biocompatible and bioresorbable, with a controllable degradation and resorption rate to match tissue growth. However, in vivo scaffold degradation and tissue ingrowth processes are not yet fully understood. Unfortunately, current analysis methods require animal sacrifice and scaffold destruction for the quantification of scaffold degradation and cannot monitor the situation in real time. In this study, Cy3, a fluorescent dye, was used for visualizing PCP and a real-time degradation profile was obtained quantitatively by a non-invasive method using an imaging system in which the reduction in fluorescence intensity depended on PCP scaffold degradation. Real-time PCP scaffold degradation was confirmed through changes in the volume and morphology of the scaffold using micro-computed tomography and microscopy. Our results suggest that extracellular matrix degradation was induced by collagen degradation because of the binding between Cy3 and collagen. This non-invasive real-time monitoring system for scaffold degradation will increase our understanding of in vivo matrix and/or scaffold degradation.

Fabrication of an osteochondral graft with using a solid freeform fabrication system

Current approaches for the engineering of osteochondral grafts are associated with poor tissue formation and compromised integration at the interface between the cartilage and bone layers. Many researchers have attempted to provide osteochondral grafts of combined cartilage and bone for osteochondral repair to help overcome the limitations of standard procedures. Solid freeform fabrication is recognized as a promising tool for creating tissue engineering scaffolds due to advantages such as superior interconnectivity and a highly porous structure. This study aimed to develop a three-dimensional plotting system to enable the manufacturing of a biphasic graft consisting cartilage and subchondral bone for application to osteochondral defects. The material advantages of both synthetic (poly L lactide-co-polyglycolide) and natural (alginate) polymers were combined for a supporting frame and cell printing. Specifically, in order to promote the maturity of the osteochondral graft in our study, cartilage-derived ECM (cECM) or hydroxyapatate (HA) substances blended with alginate was plotted together with human fetal cartilage-derived progenitor cells in the cartilage or subchondral bone layer under a multi-nozzle deposition system. Notably, a plotted biphasic graft shows good integration between cartilage and subchondral bone layers without structural separation. Furthermore, the non-toxicity of the cECM and HA substances were proved from a live/dead assay of plotted cell-laden alginate. A fabricated osteochondral graft with cECM and HA substances showed dominant cartilage and bone tissue formation in a differentiation assay. Future studies should be done to modify the alginate physical properties for long-lasting structural stability.

The Degeneration of Meniscus Roots Is Accompanied by Fibrocartilage Formation, Which May Precede Meniscus Root Tears in Osteoarthritic Knees

Background: Fibrocartilage metaplasia in tendons and ligaments is an adaptation to compression as well as a pathological feature during degeneration. Medial meniscus posterior roots are unique ligaments that resist multidirectional forces, including compression. Purpose: To characterize the degeneration of medial meniscus posterior root tears in osteoarthritic knees, with an emphasis on fibrocartilage and calcification. Study Design: Cross-sectional study; Level of evidence, 3. Methods: Samples of medial meniscus posterior roots were harvested from cadaveric specimens and patients during knee replacement surgery and grouped as follows: normal reference, no tear, partial tear, and complete tear. Degeneration was analyzed with histology, immunohistochemistry, and real-time polymerase chain reaction. Uniaxial tensile tests were performed on specimens with and without fibrocartilage. Quantifiable data were statistically analyzed by the Kruskal-Wallis test with the Dunn comparison test. Results: Thirty, 28, and 42 samples harvested from 99 patients were allocated into the no tear, partial tear, and complete tear groups, respectively. Mean modified Bonar tendinopathy scores for each group were 3.97, 9.31, and 14.15, respectively, showing a higher degree of degeneration associated with the extent of the tear (P < .05 for all groups). The characterization of root matrices revealed an increase in fibrocartilage according to the extent of the tear. Tear margins revealed fibrocartilage in 59.3% of partial tear samples and 76.2% of complete tear samples, with a distinctive cleavage-like shape. Root tears with a similar shape were induced within fibrocartilaginous areas during uniaxial tensile testing. Even in the no tear group, 56.7% of samples showed fibrocartilage in the anterior margin of the root, adjacent to the meniscus. An increased stained area of calcification and expression of the ectonucleotide pyrophosphatase/phosphodiesterase 1 gene were observed in the complete tear group compared with the no tear group (P < .0001 and P = .24, respectively). Conclusion: Fibrocartilage and calcification increased in medial meniscus posterior roots, associated with the degree of the tear. Both findings, which impair the ligament’s resistance to tension, may play a pivotal role during the pathogenesis of degenerative meniscus root tears in osteoarthritic knees. Fibrocartilage and calcification may be useful as diagnostic markers as well as markers of degeneration, which may aid in determining the treatment modality in meniscus root tears. The presence of fibrocartilage in intact roots may suggest an impending tear in osteoarthritic knees.

Polyethylene Wear Particles Play a Role in Development of Osteoarthritis via Detrimental Effects on Cartilage, Meniscus, and Synovium

OBJECTIVE While ultra-high molecular weight polyethylene (UHMWPE) wear particles are known to cause periprosthetic osteolysis, its interaction with other intra-articular tissues in the case of partial joint arthroplasties is not well understood. We hypothesized that UHMWPE particles per se would interact with intra-articular tissue, which by acting as inflammatory reservoirs, would subsequently induce osteoarthritic (OA) changes. Our goal was to assess the inflammatory response, phagocytic activity, as well as apoptosis of intra-articular cells in the presence of UHMWPE particles in vitro, and the in vivo response of those tissues after intra-articular injection of particles in a murine model. DESIGN Three cell types were used for the in vitro study; chondrocytes, meniscal fibrochondrocytes, and synoviocytes. Each cell type was cultured with two different concentrations of UHMWPE particles. Pro-inflammatory cytokine production, phagocytosis, and apoptosis were analyzed. In vivo experiments were done by injecting two concentrations of UHMWPE particles into normal and murine OA model knee joints. RESULTS In vitro experiments showed that UHMWPE particles increase pro-inflammatory cytokine and mediator (IL-1β, IL-6, TNF-α, Nitric Oxide, and Prostaglandin E2) production, phagocytosis of particles, and apoptosis in all cell types. In vivo experiment showed degeneration of cartilage and meniscus, as well as synovitis after particle injection. CONCLUSIONS UHMWPE wear particles per se exert detrimental effects in cartilage, synovium, and meniscus of the knee joint resulting in pro-inflammatory cytokine release, phagocytosis of particles and apoptosis. Particles induced and exacerbated OA changes in a murine model.

Repair of partial thickness cartilage defects using cartilage extracellular matrix membrane-based chondrocyte delivery system in human Ex Vivo model

Treatment options for partial thickness cartilage defects are limited. The purpose of this study was to evaluate the efficacy of the chondrocyte-seeded cartilage extracellular matrix membrane in repairing partial thickness cartilage defects. First, the potential of the membrane as an effective cell carrier was investigated. Secondly, we have applied the chondrocyte-seeded membrane in an ex vivo, partial thickness defect model to analyze its repair potential. After culture of chondrocytes on the membrane in vitro, cell viability assay, cell seeding yield calculation and cell transfer assay were done. Cell carrying ability of the membrane was also tested by seeding different densities of cells. Partial defects were created on human cartilage tissue explants. Cell-seeded membranes were applied using a modified autologous chondrocyte implantation technique on the defects and implanted subcutaneously in nude mice for 2 and 4 weeks. In vitro data showed cell viability and seeding yield comparable to standard culture dishes. Time dependent cell transfer from the membrane was observed. Membranes supported various densities of cells. Ex vivo data showed hyaline-like cartilage tissue repair, integrated on the defect by 4 weeks. Overall, chondrocyte-seeded cartilage extracellular membranes may be an effective and feasible treatment strategy for the repair of partial thickness cartilage defects.

Extracellular Matrix (ECM) Multilayer Membrane as a Sustained Releasing Growth Factor Delivery System for rhTGF-β3 in Articular Cartilage Repair

Recombinant human transforming growth factor beta-3 (rhTGF-β3) is a key regulator of chondrogenesis in stem cells and cartilage formation. We have developed a novel drug delivery system that continuously releases rhTGF-β3 using a multilayered extracellular matrix (ECM) membrane. We hypothesize that the sustained release of rhTGF-β3 could activate stem cells and result in enhanced repair of cartilage defects. The properties and efficacy of the ECM multilayer-based delivery system (EMLDS) are investigated using rhTGF-β3 as a candidate drug. The bioactivity of the released rhTGF-ß3 was evaluated through chondrogenic differentiation of mesenchymal stem cells (MSCs) using western blot and circular dichroism (CD) analyses in vitro. The cartilage reparability was evaluated through implanting EMLDS with endogenous and exogenous MSC in both in vivo and ex vivo models, respectively. In the results, the sustained release of rhTGF-ß3 was clearly observed over a prolonged period of time in vitro and the released rhTGF-β3 maintained its structural stability and biological activity. Successful cartilage repair was also demonstrated when rabbit MSCs were treated with rhTGF-β3-loaded EMLDS ((+) rhTGF-β3 EMLDS) in an in vivo model and when rabbit chondrocytes and MSCs were treated in ex vivo models. Therefore, the multilayer ECM membrane could be a useful drug delivery system for cartilage repair.