Pan-Pan Chong

Human peripheral blood derived mesenchymal stem cells demonstrate similar characteristics and chondrogenic differentiation potential to bone marrow derived mesenchymal stem cells

The use of mesenchymal stem cells (MSCs) for cartilage repair has generated much interest owing to their multipotentiality. However, their significant presence in peripheral blood (PB) has been a matter of much debate. The objectives of this study are to isolate and characterize MSCs derived from PB and, compare their chondrogenic potential to MSC derived from bone marrow (BM). PB and BM derived MSCs from 20 patients were isolated and characterized. From 2 ml of PB and BM, 5.4 ± 0.6 million and 10.5 ± 0.8 million adherent cells, respectively, were obtained by cell cultures at passage 2. Both PB and BM derived MSCs were able to undergo tri‐lineage differentiation and showed negative expression of CD34 and CD45, but positively expressed CD105, CD166, and CD29. Qualitative and quantitative examinations on the chondrogenic potential of PB and BM derived MSCs expressed similar cartilage specific gene (COMP) and proteoglycan levels, respectively. Furthermore, the s‐GAG levels expressed by chondrogenic MSCs in cultures were similar to that of native chondrocytes. In conclusion, this study demonstrates that MSCs from PB maintain similar characteristics and have similar chondrogenic differentiation potential to those derived from BM, while producing comparable s‐GAG expressions to chondrocytes.

Treatment Outcomes of Alginate-Embedded Allogenic Mesenchymal Stem Cells Versus Autologous Chondrocytes for the Repair of Focal Articular Cartilage Defects in a Rabbit Model:

Background: Mesenchymal stem cells (MSCs) represent a promising alternative form of cell-based therapy for cartilage injury. However, the capacity of MSCs for chondrogenesis has not been fully explored. In particular, there is presently a lack of studies comparing the effectiveness of MSCs to conventional autologous chondrocyte (autoC) treatment for regeneration of full-thickness cartilage defects in vivo. Hypothesis: Treatment with allogenic undifferentiated MSCs (alloMSCs) results in superior cartilage tissue regeneration profiles when compared with autoC for repair of focal articular cartilage defects. Study Design: Controlled laboratory study. Methods: Full-thickness articular cartilage defects were created on the weightbearing surface of the medial femoral condyles in both knees of New Zealand White rabbits (N = 30). Six weeks after the defect was induced, the right knee was treated with either alloMSCs (n = 12) or autoC (n = 18), while the left knee remained untreated (control). The rabbits were sacrificed at 6 months after treatment for assessment of cartilage tissue regeneration, which included the Brittberg morphologic score, histologic grading by O’Driscoll score, and quantitative analysis of glycosaminoglycans per total protein content. Results: Apart from significantly higher Brittberg scores in the alloMSC treatment group (8.8 ± 0.8) versus the autoC treatment group (6.6 ± 0.8) (P = .04), both treatments showed similar cartilage regenerative profiles. All outcome measures were significantly higher in the treatment groups compared with their respective controls (P < .05). Conclusion: AlloMSCs have similar effectiveness as autoC for repair of focal cartilage defects. Both treatments resulted in superior tissue regeneration compared with untreated defects. Clinical Relevance: The results have an implication of supporting the potential use of MSCs for cartilage repair after sports injuries or diseases, in view of similar efficacy but less patient morbidity and potential cost savings as compared with conventional autoC therapy.

Chondrogenic potential of physically treated bovine cartilage matrix derived porous scaffolds on human dermal fibroblast cells.

Extracellular matrices have drawn attention in tissue engineering as potential biomaterials for scaffold fabrication because of their bioactive components. Noninvasive techniques of scaffold fabrication and cross-linking treatments are believed to maintain the integrity of bioactive molecules while providing proper architectural and mechanical properties. Cartilage matrix derived scaffolds are designed to support the maintenance of chondrocytes and provide proper signals for differentiation of chondroinducible cells. Chondroinductive potential of bovine articular cartilage matrix derived porous scaffolds on human dermal fibroblasts and the effect of scaffold shrinkage on chondrogenesis were investigated. An increase in sulfated glycosaminoglycans production along with upregulation of chondrogenic genes confirmed that physically treated cartilage matrix derived scaffolds have chondrogenic potential on human dermal fibroblasts.

Characterization of undifferentiated human bone marrow and blood derived mesenchymal stem cells and their potential for chondrogenic differentiation

In recent years, the potential of cartilage tissue engineering techniques employing mesenchymal stem cells (MSCs) to repair damaged human cartilage and defects has generated much interest. Traditionally, sources of MSCs included patient’s own bone marrow. However, little have been reported on adult peripheral blood (PB) as a source of MSCs, which has been a subject of much debate amongst scientists owing to its extremely low density in PB and the difficulties associated with extracting MSCs from PB. The objectives of this study were to isolate MSCs derived from bone marrow and peripheral blood as a source and to assess their potential to undergo in vitro chondrogenesis using a biocompatible three-dimensional scaffold. In defining MSCs, the cells isolated from its source must meet these 3 criteria: (i) adherence to plastic when maintained in culture; (ii) positive expression of several antigens such as CD29, CD105, CD166; (iii) ability to differentiate into osteoblasts, adipocytes & chondrocytes under in vitro inductive conditions. PB samples (2 ml) were collected and mononuclear cells extracted and separated using Ficoll–Paque PLUS via centrifugation. Subsequently, suspended cells were removed after 5 days of culture, and adherent cells left to grow. Cells were detached upon reaching 80-90% confluence and sub-cultured up to 4 passages prior to further experiments. MSC antigens were recognized by monoclonal antibodies CD29, CD105 and CD166. To distinguish MSCs from hematopoietic stem cells, CD34 surface markers were used as negative controls. The characterized human blood-derived progenitor cells were cultured in three-dimensional alginate scaffolds using chondrogenic induction medium to promote chondrogenesis. Chondrogenesis was quantitated by sulphated glycosaminoglycan (S-GAG) production measured by 1,9-dimethylmethylene blue (DMMB) assay. Furthermore, chondrogenic-MSCs were examined and histologically compared using Safranin O staining to that of human chondrocytes as a means to determine chondrogenic transformation. Gene expression analysis was carried out by reverse transcriptase-polymerase chain reaction (RT-PCR) of differentiated human blood-derived progenitor cells using chondrocyte (cartilage cell)-specific phenotypic markers. The results showed that the cell derived in our processing technique share similar characteristics with adult MSCs and chondrocytes. Induction of chondrogenesis has been demonstrated in human blood-derived progenitor cells, which could provide a ready source of chondrocytes for engineering biological therapies. In the practical sense, PB isolation would prove to be less invasive, less expensive and less traumatic for patients to undergo therapy as compared to the 2-stage procedure of current available tissue engineering technique.

Factors Influencing the Successful Isolation and Expansion of Aging Human Mesenchymal Stem Cells

Abstract Most studies highlight mesenchymal stem cells (MSCs) extracted primarily from bone marrow (BM), very few report the use of peripheral blood (PB), often due to the associated low seeding density and difficulties with extraction techniques. As ageing populations are becoming more predominant globally, together with escalating demands for MSC transplantation and tissue regeneration, obtaining quality MSCs suitable for induced differentiation and biological therapies becomes increasingly important. In this study, BM and PB were obtained from elderly patients and extracted MSCs grown in vitro to determine their successful isolation and expansion. Patients’ socio-demographic background and other medical information were obtained from medical records. Successful and failed cultures were correlated with key demographic and medical parameters. A total of 112 samples (BM or PB) were used for this study. Of these, 50 samples (44.6%) were successfully cultured according to standardised criteria with no signs of contamination. Our comparative analyses demonstrated no statistical correlation between successful MSC cultures and any of the six demographic or medical parameters examined, including sample quantity, age, sex, race, habits and underlying comorbidities of sample donors. In conclusion, the present study demonstrates that typical demographics and comorbidities do not influence successful MSC isolation and expansion in culture.

Repair of rabbit focal articular cartilage defects with autologous chondrocytes embedded in alginate

To evaluate the ability of autologous chondrocyte transplantation to repair articular cartilage defects, autologous chondrocytes embedded in alginate beads were implanted in focal cartilage defects created in 9 New Zealand white rabbits. After 4 weeks of cartilage damage, the right knee was repaired via autologous chondrocyte-alginate constructs transplantation and the left knee was left untreated (control group). The quality of cartilage tissues of both knees was then compared at 3 months following the procedure, as reflected by the quantitative analysis of glucosaminoglycan (GAG) in the cartilage and histological examination of the tissue in accordance to the Brittberg scoring scale. Macroscopic examination showed better regeneration of the defective area following chondrocyte-alginate transplantation repair compared to the non-treated site. Biochemical analysis revealed significantly higher cellular expression of GAG in the treated knee as compared to the non-treated knee [1.12 ± 0.48 µg GAGs / mg protein vs. 0.81 ± 0.17 µg GAGs / mg protein, respectively; p=0.008]. The mean Brittberg scores was significantly higher in the treated knee as compared to control knee [6.00 ± 1.23 vs. 1.89 ± 1.54; p=0.007]. This result can be explained by the fact that chondrocytes cultured in alginate gel beads retained their ability to synthesize cartilage-specific molecules. The alginate beads were perfectly biocompatible with chondrocytes and surrounding cartilage tissue. These findings also indicated that chondrocyte-alginate transplantation has shown enhanced repair results compared to the nontreated measures.