Clinical use of stem cells in orthopaedics

Stem cell research arose from the need to explore new therapeutic possibilities for intractable and lethal diseases. Although musculoskeletal disorders are basically nonlethal, their high prevalence and relative ease of performing clinical trials have facilitated the clinical application of stem cells in this field. However, few reliable clinical studies have been published, despite the plethora of in vitro and preclinical studies in stem cell research for regenerative medicine in the musculoskeletal system. Stem cell therapy can be applied locally for bone, cartilage and tendon regeneration. Candidate disease modalities in bone regeneration include large bone defects, nonunion of fractures, and osteonecrosis. Focal osteochondral defect and osteoarthritis are current targets for cartilage regeneration. For tendon regeneration, bone-tendon junction problems such as rotator cuff tears are hot topics in clinical research. To date, the literature supporting stem cell-based therapies comprises mostly case reports or case series. Therefore, high-quality evidence, including from randomised clinical trials, is necessary to define the role of cell-based therapies in the treatment of musculoskeletal disorders. It is imperative that clinicians who adopt stem cell treatment into their practices possess a good understanding of the natural course of the disease. It is also highly recommended that treating physicians do not thrust aside the concomitant use of established measures until stem cell therapy is evidently proved worthy in terms of efficacy and cost. The purpose of this review is to summarise on the current status of stem cell application in the orthopaedic field along with the author's view of future prospects.

Treatment of FGF-2 on stem cells from inflamed dental pulp tissue from human deciduous teeth.

OBJECTIVE: The purposes of this study were to isolate and characterize stem cells from inflamed pulp tissue of human functional deciduous teeth (iSHFD) and to evaluate the influence of fibroblastic growth factor-2 (FGF-2) on the regenerative potential. MATERIALS AND METHODS: We successfully isolated mesenchymal stem cells (MSCs) from the inflamed dental pulp tissue of human deciduous teeth and demonstrated that their regenerative potential could be enhanced by the application of FGF-2 (20 ng ml(-1)) during ex vivo expansion. Isolated stem cells expanded in FGF-2 were characterized using a colony-forming assay, proliferation, migration, in vitro differentiation, in vivo ectopic transplantation assay, and gene expression profiling. RESULTS: MSCs isolated from the inflamed pulp tissue of functional deciduous teeth potentially possess the qualities of those from human exfoliated deciduous teeth. FGF-2 applied to iSHFD during expansion enhanced the colony-forming efficiency of these cells, increased their proliferation and migration potential, and reduced their differentiation potential in vitro. However, the ectopic transplantation of iSHFD/FGF-2 in vivo increased the formation of dentin-like material. CONCLUSION: FGF-2 expansion of stem cells from inflamed pulp tissues of human deciduous teeth can be a good source of stem cells for future clinical applications and a novel way of using discarded inflamed tissues.

Dose- and time-dependent effects of recombinant human bone morphogenetic protein-2 on the osteogenic and adipogenic potentials of alveolar bone-derived stromal cells.

BACKGROUND AND OBJECTIVE: Recombinant human bone morphogenetic protein-2 (rhBMP-2) is a well-known growth factor that can induce robust bone formation, and recent studies have shown that rhBMP-2-induced osteogenesis is closely related to adipogenesis. The aim of the present study was to determine the dose- and time-dependent effects of rhBMP-2 on the osteogenic and adipogenic differentiation of human alveolar bone-derived stromal cells (hABCs) in vivo and in vitro. MATERIAL AND METHODS: hABCs were isolated and cultured, and then transplanted using a carrier treated either with or without rhBMP-2 (100 µg/mL) into an ectopic subcutaneous mouse model. Comprehensive histologic and histometric analyses were performed after an 8-wk healing period. To further understand the dose-dependent (0, 10, 50, 200, 500 and 1000 ng/mL) and time-dependent (0, 3, 5, 7 and 14 d) effects of rhBMP-2 on osteogenic and adipogenic differentiation, in vitro osteogenic and adipogenic differentiation of hABCs were evaluated, and the expression of related mRNAs, including those for alkaline phosphatase, osteocalcin, bone sialoprotein, peroxisome-proliferator-activated receptor gamma-2 and lipoprotein lipase, were assessed using quantitative RT-PCR. RESULTS: rhBMP-2 significantly promoted the osteogenic and adipogenic differentiation of hABCs in vivo, and gradually increased both the osteogenic and adipogenic potential in a dose- and time-dependent manner with minimal deviation in vitro. The expression of osteogenesis- and adipogenesis-associated mRNAs were concomitantly up-regulated by rhBMP-2. CONCLUSION: The findings of the present study showed that rhBMP-2 significantly enhanced the adipogenic as well as the osteogenic potential of hABCs in dose- and time-dependent manner. The control of adipogenic differentiation of hABCs should be considered when regenerating the alveolar bone using rhBMP-2.

Effect of humoral factors from hPDLSCs on the biologic activity of hABCs.

OBJECTIVE: The human periodontal ligament stem cells (hPDLSCs) and human alveolar bone-derived stromal cells (hABCs) seem to be closely involved in the maintenance of alveolar bone in an anatomically indirect manner; however, there is little study on this matter. Therefore, the effect of hPDLSCs on the osteoclastogenic, osteogenic, and adipogenic differentiation of hABCs was evaluated, focusing on the humoral factors released by hPDLSCs. MATERIALS AND METHODS: Human periodontal ligament stem cells and hABCs were isolated and characterized. hPDLSCs were indirectly cocultured to observe the in vitro effect of humoral factors released from hPDLSCs on the osteoclastogenic, osteogenic, and adipogenic differentiation of hABCs. Human gingival fibroblasts (hGFs) were utilized as positive control. RESULTS: Isolated cells demonstrated the presence of stem cells within. Indirect coculture of hPDLSCs greatly inhibited osteoclastogenesis by hABCs. Osteogenesis/adipogenesis of hABCs was also inhibited by indirect coculture with hPDLSC. The magnitude of regulatory effect from hPDLSCs was significantly greater than that of hGFs. CONCLUSIONS: Humoral factors released from hPDLSCs seemed to modulate the differentiation of hABCs, and the osteoclastogenic, osteogenic, and adipogenic differentiation of hABCs was all inhibited, suggesting the potential role of hPDLSCs in the maintenance of the alveolar bone.

The dynamic healing profile of human periodontal ligament stem cells: histological and immunohistochemical analysis using an ectopic transplantation model.

BACKGROUND AND OBJECTIVE: Human periodontal ligament stem cells (hPDLSCs) have been reported to play the pivotal role in periodontal regeneration. However, the dynamic cellular healing process initiated by hPDLSCs still remains to be elucidated. In the present study, the sequence of regeneration by hPDLSCs was assessed using histological and immunohistochemical observation in an ectopic transplantation model, which is a well-standardized assessment tool that excludes the innate healing factors from the animals. MATERIAL AND METHODS: Human periodontal ligament stem cells that were isolated and characterized from teeth (n=12) extracted for the purpose of orthodontic treatment were transplanted with carriers into ectopic subcutaneous pouches in immunocompromised mice (n=20). Animals were killed after several different healing periods: 3 d (n=4), 1 (n=4), 2 (n=4), 4 (n=4) and 8 wk (n=4). Histological analysis for regenerated tissues formed by hPDLSCs was conducted using hematoxylin and eosin, Masson's trichrome and picrosirius red staining. In addition, immunohistochemical staining was performed to observe the sequential expression of osteogenic/cementogenic and periodontal ligament tissue-specific markers associated with periodontal regeneration. RESULTS: The whole healing process by transplanted hPDLSCs could be broadly divided into four distinctive phases. In the first phase, proliferated hPDLSCs migrated evenly all over the carrier, and collagenous tissues appeared in the form of amorphous collagen matrices. In the second phase, collagen fibers were well arranged among the carriers, and cementoid-like tissues were observed. In the third phase, the formation of mature collagen fibers, resembling Sharpey's fibers, was associated with active mineralization of cementum-like tissues, and in the fourth phase, the maturation of cementum-like tissues was observed on carrier surfaces. Various osteogenic/cementogenic markers related to the regeneration processes were expressed in a well-orchestrated time order. Interestingly, well-organized cementum-like and periodontal ligament fiber-like tissues and cells with early and late osteogenic/cementogenic markers were frequently observed in the secluded area of carrier surfaces. We termed this area the cell-rich zone. CONCLUSION: The results from this study clearly demonstrated the sequential histological changes during periodontal tissue regeneration by hPDLSCs. Understanding of this process would potentially enable us to develop better cell-based treatment techniques.

Electroporation-mediated gene transfer of SOX trio to enhance chondrogenesis in adipose stem cells.

OBJECTIVE: The aim of the present study was to determine if the electroporation-mediated gene transfer of SOX trio enhances the chondrogenic potential of adipose stem cells (ASCs). DESIGN: ASCs were transfected with SOX trio genes using an electroporation technique and cultured for 3 weeks. The pellets were analyzed for DNA and glycosaminoglycan (GAG) analysis, and the gene and protein expression of SOX-5, SOX-6, SOX-9, type 1 collagen (COL1Al), type 2 collagen (COL2Al) and type 10 collagen (COL10A1) using real-time PCR and Western blot analysis. Further in vivo studies were carried out by subcutaneous transplantation of pellets in severe combined immunodeficiency (SCID) mice for 3 weeks. RESULTS: The gene transfer efficiency was high (approximately 70%). Transfected ASCs showed high expression of corresponding genes after 21 days, and each SOX protein was detected in ASCs transfected with the corresponding gene. The chondrogenic differentiation of ASCs, as demonstrated by GAG levels and Safranin-O staining, showed significant enhancement when SOX trio were co-transfected, while subsets with single gene transfer of SOX-5, -6, or -9 did not show significant elevation. SOX trio co-transfection enhanced COL2A1 mRNA, but did not increase COL1A1 and COL10A1 mRNA. Type II collagen protein dramatically increased, and type X collagen decreased with co-transfection of the SOX trio. When pellets were implanted in the subcutaneous pouch of SCID mice for 3 weeks, ASCs co-transfected with SOX trio demonstrated abundant proteoglycan, significantly reduced mineralization. CONCLUSION: The electroporation-mediated transfection of SOX trio greatly enhances chondrogenesis from ASCs, while decreasing hypertrophy.

Proximal hip geometry and hip fracture risk assessment in a Korean population.

UNLABELLED: The association between proximal femoral geometry and hip fracture risk were investigated. The risk of intertrochanteric fractures increased 1.64-fold and 2.32-fold with 1 standard deviation (sd) increase of hip axis length and neck-shaft angle, respectively, while the risk of femur neck fracture 2.03-fold with 1 sd decrease in femoral head offset. INTRODUCTION: The purpose of this study was to determine the association between proximal femoral geometry (PFG) and the risk of hip fracture in femur neck (FN) and intertrochanteric (IT) fractures in a Korean population. METHODS: The study included 151 patients (57 patients with IT fractures, 43 patients with FN fractures, and 51 control patients). Data on BMD, PFG parameters (hip axis length [HAL], neck-shaft angle [NSA], neck length, femoral head offset, neck diameter, shaft diameter (SD), and demographics [age, gender, height, and body weight]) were collected. Descriptive statistics and odds ratios of PFG parameters corrected with demographic variables were obtained using logistic regressions. RESULTS: HAL (p = 0.046) and NSA (p = 0.003) were significantly greater in the patients with IT fracture than in the control patients, while neither parameter was significantly greater in patients with FN fractures than the control patients. The femoral head offset was significantly shorter in the patients with FN fractures (p = 0.003) compared with the control patients. In patients with IT fractures, the fracture risk increased 1.64-fold (p = 0.048) with a 1 sd increase of the HAL, while it increased 2.32-fold (p = 0.003) with a 1 sd increase of the NSA. In FN fractures, the fracture risk increased 2.03-fold (p = 0.012) with a 1 sd decrease in femoral head offset. CONCLUSIONS: Our study showed that some PFG parameters as well as BMD values predict hip fractures in a Korean population, and their evaluation may be useful in the understanding of the biomechanics of hip fractures.

Radiological joint space width in the clinically normal hips of a Korean population.

OBJECTIVE: The purpose of this paper was to investigate the association of the joint space width (JSW) of the hip with radiologically observed hip deformity, the anthropological features and aging in a clinically asymptomatic Korean population. DESIGN: 428 consecutive patients who were without clinical evidence of hip osteoarthritis (OA) and who underwent supine anteroposterior (AP) pelvic radiography for hip contusion or a routine health check were analyzed for the relation of joint space narrowing to the center-edge (CE) angle, the acetabular depth, the head-neck ratio, the neck-shaft angle, the pelvic width, the height, the body mass index (BMI), gender and age. RESULTS: The CE angle was inversely associated with the superomedial JSW and the superolateral JSW. The acetabular depth was positively associated with superomedial JSW. A decreased head-neck ratio and the neck-shaft angle were not associated with the superomedial or superolateral JSW. The height was positively associated with an increased superomedial JSW, but not with the superolateral JSW. The BMI and increased age were positively associated with the superolateral JSW, but not with the superomedial JSW. CONCLUSION: Our study showed that the CE angle was the single constant radiological parameter that was inversely related to the JSW of hip joints. Further, the height was positively related to the superomedial JSW while the BMI was positively related to the superolateral JSW. The normal aging process was not associated with joint space narrowing of the hip joint.

Suppressive effects of interleukin-4 and interleukin-10 on the production of proinflammatory cytokines induced by titanium-alloy particles.

We investigated the effects of two anti-inflammatory cytokines, interleukin (IL)-4 and IL-10, on the production of two proinflammatory cytokines, tumor necrosis factor (TNF)-alpha and IL-6, induced by titanium-alloy (Ti6Al4V) particles. Human monocytes isolated from the peripheral blood of six healthy donors were cultured and exposed to retrieved titanium-alloy particles, together with IL-4 or IL-10. Enzyme-linked immunosorbent assay and reverse transcription-polymerase chain reaction showed that both IL-4 and IL-10 significantly reduced the production of TNF-alpha and IL-6. The level of TNF-alpha decreased by 74% with IL-4 and 56% with IL-10. The level of IL-6 decreased by 55% with IL-4 and by 66% with IL-10. IL-4 had more consistent suppressive effect on TNF-alpha production. The results from this study provide in vitro evidence of the possible utility of IL-4 and IL-10 in suppression of particle-induced activation of macrophages.

Repair of cartilage defect in the rabbit with cultured mesenchymal stem cells from bone marrow.

In 16 mature New Zealand white rabbits mesenchymal stem cells were aspirated from the bone marrow, cultured in monolayer and implanted on to a full-thickness osteochondral defect artificially made on the patellar groove of the same rabbit. A further 13 rabbits served as a control group. The rabbits were killed after 14 weeks. Healing of the defect was investigated histologically using haematoxylin and eosin and Safranin-O staining and with immunohistochemical staining for type-II collagen. We also used a reverse transcription-polymerase chain reaction (RT-PCR) to detect mRNA of type-I and type-II collagen. The semiquantitative histological scores were significantly higher in the experimental group than in the control group (p < 0.05). In the experimental group immunohistochemical staining on newly formed cartilage was more intense for type-II collagen in the matrix and RT-PCR from regenerated cartilage detected mRNA for type-II collagen in mature chondrocytes. These findings suggest that repair of cartilage defects can be enhanced by the implantation of cultured mesenchymal stem cells.

Degeneration of the acetabular cartilage in osteonecrosis of the femoral head: histopathologic examination of 15 hips.

Acetabular cartilage with subchondral bone was taken from the superior dome from 15 hips of 13 patients undergoing total hip arthroplasty due to osteonecrosis of the femoral head. The mean age of the patients was 40 years. There were 10 hips ARCO stage IIIA, and 5 hips stage IIIB. 3 of the cases were mild, 12 moderate, and 1 had severe arthrosis. The degree of collapse of the femoral head was significantly related to the degeneration of the acetabular cartilage on histological examination. Our observations support the view that patients with an ARCO Stage III hip do not benefit from head-preserving procedures. They may also explain why bipolar prosthesis gives poorer results than total hip arthroplasty, in cases of osteonecrosis of the femoral head.