Norihiko Sugita

Next Generation Mesenchymal Stem Cell (MSC)-Based Cartilage Repair Using Scaffold-Free Tissue Engineered Constructs Generated with Synovial Mesenchymal Stem Cells.

Because of its limited healing capacity, treatments for articular cartilage injuries are still challenging. Since the first report by Brittberg, autologous chondrocyte implantation has been extensively studied. Recently, as an alternative for chondrocyte-based therapy, mesenchymal stem cell–based therapy has received considerable research attention because of the relative ease in handling for tissue harvest, and subsequent cell expansion and differentiation. This review summarizes latest development of stem cell therapies in cartilage repair with special attention to scaffold-free approaches.

Morphological Observations of Mesenchymal Stem Cell Adhesion to a Nanoperiodic-Structured Titanium Surface Patterned Using Femtosecond Laser Processing

It is known that the adhesive and anisotropic properties of cell-derived biomaterials are affected by micro- or nanoscale structures processed on culture surfaces. In the present study, the femtosecond laser processing technique was used to scan a laser beam at an intensity of approximately the ablation threshold level on a titanium surface for nanoscale processing. Microscopy observation revealed that the processed titanium exhibited a periodic-patterned groove structure at the surface; the width and depth of the groove were 292 ±50 and 99 ±31 nm, respectively, and the periodic pitch of the groove was 501 ±100 nm. Human synovium-derived mesenchymal stem cells were cultured on the surface at a cell density of 3.0×103 cells/cm2 after 4 cell passages. For comparison, the cells were also cultured on a nonprocessed titanium surface under the condition identical to that of the processed surface. Results revealed that the duration for cell attachment to the surface was markedly reduced on the processed titanium as compared with the nonprocessed titanium. Moreover, on the processed titanium, cell extension area significantly increased while cell orientation was aligned along the direction of the periodic grooves. These results suggest that the femtosecond laser processing improves the adhesive and anisotropic properties of cells by producing the nanoperiodic structure on titanium culture surfaces.

Optimization of human mesenchymal stem cell isolation from synovial membrane: Implications for subsequent tissue engineering effectiveness

Synovium-derived mesenchymal stem cells (SDMSCs) are one of the most suitable sources for cartilage repair because of their chondrogenic and proliferative capacity. However, the isolation methods for SDMSCs have not been extensively characterized. Thus, our aim in this study was to optimize the processes of enzymatic isolation followed by culture expansion in order to increase the number of SDMSCs obtained from the original tissue. Human synovium obtained from 18 donors (1.5 g/donor) was divided into three aliquots. The samples were minced and subjected to collagenase digestion, followed by different procedures: Group 1, Tissue fragments were removed by filtering followed by removing floating tissue; Group 2, No filtering. Only floating fragments were removed; Group 3, No fragments were removed. Subsequently, each aliquot was sub-divided into two density subgroups with half. In Group 1, the cell-containing media was plated either at high (5000 cells/cm2) or low density (1000 cells/cm2). In Groups 2 and 3, the media containing cells and tissue was plated onto the same number of culture dishes as used in Group 1, either at high or low density. At every passage, the cells plated at high density were consistently re-plated at high and those plated at low density were likewise. The expanded cell yields at day 21 following cell isolation were calculated. These cell populations were then evaluated for their osteogenic, adipogenic, and chondrogenic differentiation capabilities. The final cell yields per 0.25 g tissue in Group 1 were similar at high and low density, while those in Groups 2 and 3 exhibited higher when cultured at low density. The cell yields at low density were 0.7 ± 1.2 × 107 in Group 1, 5.7 ± 1.1 × 107 in Group 2, 4.3 ± 1.2 × 107 in Group 3 (Group 1 vs Groups 2 and 3, p < 0.05). In addition, the cells obtained in each low density subgroup exhibited equivalent osteogenic, adipogenic, and chondrogenic differentiation. Thus, it was evident that filtering leads to a loss of cells and does not affect the differentiation capacities. In conclusion, exclusion of a filtering procedure could contribute to obtain higher number of SDMSCs from synovial membrane without losing differentiation capacities.

Repairing Osteochondral Defects of Critical Size Using Multiple Costal Grafts An Experimental Study

Objective: To investigate the feasibility of repairing osteochondral defects of critical size by performing mosaicplasty using multiple sliced costal cartilage grafts, which enables repair of extensively injured knees using grafts from a single rib. Design: Critical osteochondral defects were prepared on the femoral groove of skeletally mature Japanese white rabbits. Costal cartilage grafts from a single rib were harvested and sliced into multiple segments (approximately 3-5 mm in length). The defects were left untreated or repaired by performing mosaicplasty using costal cartilage grafts (with or without a longitudinal cut along the middle). At 4 and 12 weeks after transplantation, International Cartilage Repair Society macroscopic and histological grading was performed. Results: The macroscopic score and visual histological score were significantly higher in the repaired groups than in the untreated group at 4 and 12 weeks after surgery. Histological continuous integration between grafted costal cartilage and host bone was observed in both repaired groups. Conclusions: The findings suggest that costal cartilage might be a useful alternative source for chondral grafting. We were able to repair large osteochondral defects by performing mosaicplasty using multiple sliced costal cartilage grafts from a single rib.

Time-Dependent Recovery of Human Synovial Membrane Mesenchymal Stem Cell Function After High-Dose Steroid Therapy: Case Report and Laboratory Study:

Background: The use of mesenchymal stem cells from various tissue sources to repair injured tissues has been explored over the past decade in large preclinical models and is now moving into the clinic. Purpose: To report the case of a patient who exhibited compromised mesenchymal stem cell (MSC) function shortly after use of high-dose steroid to treat Bell’s palsy, who recovered 7 weeks after therapy. Study Design: Case report and controlled laboratory study. Methods: A patient enrolled in a first-in-human clinical trial for autologous implantation of a scaffold-free tissue engineered construct (TEC) derived from synovial MSCs for chondral lesion repair had a week of high-dose steroid therapy for Bell’s palsy. Synovial tissue was harvested for MSC preparation after a 3-week recovery period and again at 7 weeks after therapy. Results: The MSC proliferation rates and cell surface marker expression profiles from the 3-week sample met conditions for further processing. However, the cells failed to generate a functional TEC. In contrast, MSCs harvested at 7 weeks after steroid therapy were functional in this regard. Further in vitro studies with MSCs and steroids indicated that the effect of in vivo steroids was likely a direct effect of the drug on the MSCs. Conclusion: This case suggests that MSCs are transiently compromised after high-dose steroid therapy and that careful consideration regarding timing of MSC harvest is critical. Clinical Relevance: The drug profiles of MSC donors and recipients must be carefully monitored to optimize opportunities to successfully repair damaged tissues.

First-in-Human Pilot Study of Implantation of a Scaffold-Free Tissue-Engineered Construct Generated From Autologous Synovial Mesenchymal Stem Cells for Repair of Knee Chondral Lesions:

Background: Articular cartilage has limited healing capacity, owing in part to poor vascularity and innervation. Once injured, it cannot be repaired, typically leading to high risk for developing osteoarthritis. Thus, cell-based and/or tissue-engineered approaches have been investigated; however, no approach has yet achieved safety and regenerative repair capacity via a simple implantation procedure. Purpose: To assess the safety and efficacy of using a scaffold-free tissue-engineered construct (TEC) derived from autologous synovial membrane mesenchymal stem cells (MSCs) for effective cartilage repair. Study Design: Case series; Level of evidence, 4. Methods: Five patients with symptomatic knee chondral lesions (1.5-3.0 cm2) on the medial femoral condyle, lateral femoral condyle, or femoral groove were included. Synovial MSCs were isolated from arthroscopic biopsy specimens and cultured to develop a TEC that matched the lesion size. The TECs were then implanted into chondral defects without fixation and assessed up to 24 months postoperatively. The primary outcome was the safety of the procedure. Secondary outcomes were self-assessed clinical scores, arthroscopy, tissue biopsy, and magnetic resonance image–based estimation of morphologic and compositional quality of the repair tissue. Results: No adverse events were recorded, and self-assessed clinical scores for pain, symptoms, activities of daily living, sports activity, and quality of life were significantly improved at 24 months after surgery. Secure defect filling was confirmed by second-look arthroscopy and magnetic resonance imaging in all cases. Histology of biopsy specimens indicated repair tissue approaching the composition and structure of hyaline cartilage. Conclusion: Autologous scaffold-free TEC derived from synovial MSCs may be used for regenerative cartilage repair via a sutureless and simple implantation procedure. Registration: 000008266 (UMIN Clinical Trials Registry number).

Current Strategies in Osteochondral Repair with Biomaterial Scaffold

Osteoarthritis (OA) is a common disease defined as degenerative arthritis or joint disease involving degradation of articular cartilage and subchondral bone, and it could potentially affect the quality of life of elderly populations worldwide. The management of OA remains challenging and controversial. Although there are several clinical options for the treatment of OA, regeneration of the damaged articular cartilage has proven difficult due to the limited healing capacity. With the advancements in tissue engineering approaches including cell-based technologies and development of biomaterial scaffolds over the past decade, new therapeutic options for patients with osteochondral lesions potentially exist. This chapter will focus on the feasibility of tissue-engineered biomaterial scaffolds, which can mimic the native osteochondral complex, for osteochondral repair and highlight the recent development of these techniques toward tissue regeneration, which will contribute to osteochondral repair for the patients who are involved with an incurable OA treated by traditional procedures. Moreover, basic anatomy, strategy for osteochondral repair, and the design and fabrication methods of scaffolds as well as the choice of cells, growth factor, and materials will be discussed. Specifically, we focus on the latest preclinical animal studies using large animals and clinical trials with high clinical relevance. Accordingly, this will contribute to an understanding of the latest trends in osteochondral repair and future application of such clinical therapies in patients with OA.

37 Mechanical And Structural Properties Of Stem Cell-based Tissue Engineered Constructs (tec) Cultured With Collagen Sheets

Introduction Stem cell-based tissue engineered construct (TEC) biosynthesized from synovium-derived mesenchymal stem cells (MSCs) has a great potential for repairing and regenerating ligaments and tendons1. However, the mechanical and structural properties of the TEC were insufficient for clinical applications. A candidate solution to the problem is to promote the generation of the extracellularmatrix inthe TECusing a special culture withcollagen sheets (CS). Previous studies indicated thattheCS has an abilitytorepair fibrous tissuesin vivo4. In addition, the biosynthesis of extracellular matrix is promoted by cell culturing in the existence of collagen.2 In the present study, TECwas culturedwiththe CS, then the tensile properties of TEC/CS composite were assessed. Materials and Methods The CS wasproduced from porcine corium-derived collagen-rich tissues, through homogenization, centrifugal separation, filtering, and dehydration. It should be noted that the sheets are porous and chemical agent-free materials with collagen density between 20 and 30%. MSCswere obtained from the synovial membranes of human knee joint by means of collagenase treatment. After 5 time passages, the cells were plated at the density of 4.0 × 105 cells/cm2 on the CS (TEC/CS), and ontissue culturepolystyrenedishes (TEC). The cells were culturedto produce construct of extracellular matrix for 1, 4, and 8 weeks in the DMEM1. In addition, the CS was also immersed in the same medium without cells for 4 weeks (CS). TEC/CS, TEC, and CS weresubjected to tensiletest at arate of 0.05 mm/s in a PBS solution at 37 °C using acustom-made micro tensile tester.3 Morphological observations of the tissues were carried out using a scanning electron microscopic (SEM)and an optical microscope with hematoxylin-eosin (HE) staining. Results and discussion The tangent modulus and tensile strengthofTEC/CSgroupwere continuously increased. In addition, those of TEC/CS after 8-week cultivation were significantly higher than those of CS. The load at failure of TEC/CS group was significantly lower than those of CS and TEC groups at 1 week. However, it was increased subsequently and reached to 0.18 ± 0.05 N at 8 weeks, with significant differences vs. CS (0.07 ± 0.02 N) and TEC (0.12 ± 0.05 N) groups. Abstract 37 Figure 1 Morphological observation of TEC/CS composites (S: Surface, C: Cross section). Morphological observation indicated that CS was partially degraded with MSCs invasion into shallow CS layers for 2–4 weeks. However, newly formed fibrous tissues were observed in the CS layers at 8 weeks (Figure 1). It is suggested that these morphological changes corresponded well with the changes in mechanical and structural properties of the TEC/CS composites. References 1 Ando W, et al. Biomaterials. 2007;28:5462–5470 2 Gentleman E, et al. Biomaterials. 2003;24: 3805–3813 3 Nagai H, et al. Japanese Jounal of Clinical Biomechanics. 2006;27:89–93 [in Japanese] 4 Suzuki D, et al. abstract. Jounal of Japanese Clinical Biomechanics. 39