Rohan R. Varshney

Synovium-Derived Mesenchymal Stem Cells: A New Cell Source for Musculoskeletal Regeneration

Ever since synovium-derived mesenchymal stem cells (SMSCs) were first identified and successfully isolated in 2001, as a brand new member in MSC families, they have been increasingly regarded as a promising therapeutic cell species for musculoskeletal regeneration, particularly for reconstructions of cartilage, bones, tendons, and muscles. Besides the general multipotency in common among the MSC community, SMSCs excel other sourced MSCs in higher ability of proliferation and superiority in chondrogenesis. This review summarizes the latest advances in SMSC-related studies covering their specific isolation methodologies, biological insights, and practical applications in musculoskeletal therapeutics of which an emphasis is cast on engineered chondrogenesis.

In-vitro osteogenesis of synovium stem cells induced by controlled release of bisphosphate additives from microspherical mesoporous silica composite.

In this study, in-vitro osteogenesis was successfully induced in the highly chondrogenic synovium mesenchymal stem cells (SMSCs) by controlled release of a nitrogenous bisphosphonate additive--alendronate (AL) from a mesoporous silica (MS)-hydroxyapatite (HA) composite that was mediated in poly(lactic-co-glycolic acid) (PLGA) microspheres. This microspherical based controlled release system is constructed with three levels of degradable structures: (1) the AL drug was first hybridized with HA nanoparticles; (2) the HA-AL complexes were filled into the mesopores of MS particles by self-assembly in situ; and (3) the HA-AL-laden MS constructs (MSH-AL) were built in the bulk of PLGA microspheres. In comparison with any mono-component construct, the superiority of this multi-component system comes from two aspects of functionalities: (1) significantly greater loading capacity of the extremely hydrophilic drug-AL; and (2) better controlled profile of AL release. Based on this newly developed PLGA/MSH-AL releasing system, as recipients the SMSCs, which usually exhibit exclusively high chondrogenesis, demonstrated a strong osteogenic commitment. The results were verified by alkaline phosphatase (ALP) activity assay, calcium secretion assay, real time PCR and immunohistochemistry analysis. Considering the renewable source and high proliferative profile of SMSCs, the achievement of engineered SMSC osteogenesis with this PLGA/MSH-AL controlled release system would open a new door to major bony reparation and regeneration.

In vitro engineered cartilage using synovium-derived mesenchymal stem cells with injectable gellan hydrogels

Synovium-derived mesenchymal stem cells (SMSC), a novel line of stem cells, are regarded as a promising cell source for cartilage tissue engineering. The goal of this study was to investigate rabbit SMSC coupled with injectable gellan hydrogels for in vitro engineered cartilage. SMSC were isolated from rabbit synovial tissue, amplified to passage 4 in monolayer, and encapsulated in injectable gellan hydrogels, constructs of which were cultured in chondrogenic medium supplemented with TGF-beta1, TGF-beta3 or BMP-2 for up to 42 days. The quality of the constructs was assessed in terms of cell proliferation and chondrocytic gene/protein expression using WST-1 assay, real-time RT-PCR, biochemical analysis, histology and immunohistochemical analysis. Results indicate that the viability of SMSC in hydrogels treated with TGF-beta1, TGF-beta3 and BMP-2 remained high at culture time. The constructs formed cartilaginous tissue with the expression of chondrocytic genes (collagen type II, aggrecan, biglycan, SOX 9) and cartilaginous matrix (sulphated glycosaminoglycan and collagen) as early as 21 days in culture. Both TGF-beta1 and TGF-beta3 treated SMSC-laden hydrogels showed more chondrogenesis compared with BMP-2 treated SMSC-laden hydrogels. It demonstrates that injectable SMSC-laden gels, when treated with TGF-beta1, TGF-beta3 or BMP-2, are highly competent for in vitro engineered cartilage formation, which lays a foundation for their potential application in clinical cartilage repair.

Microsphere-based drug releasing scaffolds for inducing osteogenesis of human mesenchymal stem cells in vitro

In this study, in vitro osteogenesis was successfully achieved in human mesenchymal stem cells (hMSCs) by controlled release of the osteogenesis-inducing drugs dexamethasone, ascorbic acid (AA) and beta-glycerophosphate (GP) from poly(lactic-co-glycolic acid) (PLGA) sintered microsphere scaffolds (SMS). We investigated the osteogenesis of human MSCs (hMSCs) on dexamethasone laden PLGA-SMS (PLGA-Dex-SMS), and dexamethasone, AA and GP laden PLGA-SMS (PLGA-Com-SMS). hMSCs cultured on the microsphere systems, which act as drug release vehicles and also promote cell growth/tissue formation-displayed a strong osteogenic commitment locally. The osteogenic commitment of hMSCs on the scaffolds were verified by alkaline phosphatase (ALP) activity assay, calcium secretion assay, real-time PCR and immunohistochemistry analysis. The results indicated hMSCs cultured on PLGA-Com-SMS exhibited superior osteogenic differentiation owing to significantly high phenotypic expression of typical osteogenic genes-osteocalcin (OC), type I collagen, alkaline phosphatase (ALP), and Runx-2/Cbfa-1, and protein secretion of bone-relevant markers such as osteoclast and type I collagen when compared with PLGA-Dex-SMS. In conclusion, by promoting osteogenic development of hMSCs in vitro, this newly designed controlled release system opens a new door to bone reparation and regeneration.

Chondrogenesis of synovium-derived mesenchymal stem cells in gene-transferred co-culture system.

A co-culture strategy has been developed in this study wherein rabbit synovial mesenchymal stem cells (SMSCs) are co-cultured with growth factor (GF) transfected articular chondrocytes. Toward this end, both SMSCs and early passage rabbit articular chondrocytes that had been adenovirally transduced with transforming growth factor-beta 3 (TGF-beta3) gene were separately encapsulated in alginate beads and co-cultured in the same pool of chondrogenic medium. The chondrocytes act as transfected companion cells (TCCs) providing GF supply to induce chondrogenic differentiation of SMSCs that play the role of therapeutic progenitor cells (TPCs). Against the same TCC based TGF-beta3 release profile, the co-culture was started at different time points (Day 0, Day 10 and Day 20) but made to last for identical periods of exposure (30 days) so that the exposure conditions could be optimized in terms of initiation and duration. Transfection of TCCs prevents the stem cell based TPCs from undergoing the invasive procedure. It also prevents unpredictable complications in the TPCs caused by long-term constitutive over-expression of a GF. The adenovirally transfected TCCs exhibit a transient GF expression which results in a timely termination of GF supply to the TPCs. The TCC-sourced transgenic TGF-beta3 successfully induced chondrogenesis in the TPCs. Real-time PCR results show enhanced expression of cartilage markers and immuno/histochemical staining for Glycosaminoglycans (GAG) and Collagen II also shows abundant extracellular matrix (ECM) production and chondrogenic morphogenesis in the co-cultured TPCs. These results confirm the efficacy of directing stem cell differentiation towards chondrogenesis and cartilage tissue formation by co-culturing them with GF transfected chondrocytes.

Antisense makes sense in engineered regenerative medicine.

The use of antisense strategies such as ribozymes, oligodeoxynucleotides (ODNs) and small interfering RNA (siRNA) in gene therapy, in conjunction with the use of stem cells and tissue engineering, has opened up possibilities in curing degenerative diseases and injuries to non-regenerating organs and tissues. With their unique ability to down-regulate or silence gene expression, antisense oligonucleotides are uniquely suited in turning down the production of pathogenic or undesirable proteins and cytokines. Here, we review the antisense strategies and their applications in regenerative medicine with a focus on their efficacies in promoting cell viability, regulating cell functionalities as well as shaping an optimal microenvironment for therapeutic purposes.

Gene Transfer and Living Release of Transforming Growth Factor-β3 for Cartilage Tissue Engineering Applications

Cartilage restoration continues to present a tremendous clinical challenge due to its nonvascular nature. Many studies have demonstrated that chondrogenesis of progenitor cells can be achieved in vitro by manual dose of growth factors; however, it remains a vital difficulty in feeding growth factors to implanted therapeutic cells in vivo. Herein, we constructed recombinant adenovirus encoding human transforming growth factor-beta3 (hTGF-beta3) and practiced it in rat bone marrow-derived mesenchymal stromal cells and articular chondrocytes for cartilage regeneration. Optimal viable transduction and transgenic hTGF-beta3 production were achieved; consequently, positive expression of cartilage marker-collagen type II was enabled in the infected progenitors. We thus conclude that recombinant adenovirus encoding TGF-beta3 gene has been successfully established and validated for cartilage tissue engineering applications.

TGF-β3: A promising growth factor in engineered organogenesis

Background: Engineered organogenesis is one of the most challenging areas on the cutting edge of regenerative medicine. Growth factors can affect cell proliferation, migration and differentiation profoundly, and thus play a critical role in tissue regeneration. TGF-βs produce a wide range of effects in different cells and tissues. TGF-β3 is relatively recently discovered and studied. Objective: To provide a broader understanding of the current state of TGF-β3 in engineered osteogenesis, chondrogenesis, palate development, scar-free wound healing, odontogenesis and neurogenesis. Methods: This review summarizes studies that explore or apply TGF-β3 for organogenesis with engineering methodology and a regenerative medical perspective. Results/conclusion: TGF-β3 has proven to be a competent growth factor in engineered organogenesis in vitro. In recent years, using TGF-β3, more and more in vivo studies have yielded significant therapeutic achievements in animal models, which bear much promise for future medical application.

Sintered Microsphere Scaffolds for Controlled Release and Tissue Engineering

Scaffolds are an essential component in tissue engineering, as they provide a three-dimensional niche for cell residence and tissue regeneration (1). In recent decades, further incorporation of controlled release functionalities into newly designed scaffolds, in pursuit of better regulation and promotion of tissue development, has drawn more and more attention. Sintered microsphere scaffolds (SMSs), first developed in 2001 (2), manage to combine the excellence of controlled release functions, which are inherited from common degradable microspheres, and the talent of cell delivery, which belongs to generic porous scaffolds, so that unique superiorities are assembled together. Besides the capacity of controlled release, SMSs also exhibit excellent initial mechanical properties. The compressive strength and compressive modulus of polymerbased SMSs are comparable to those of cancellous bone (3). Currently, protocols for fabrication of SMSs via heating are based on Borden’s recipe (4) (Fig. 1). Briefly, prefabricated polymer microspheres produced by solvent evaporation technique are poured into a mold and heated to a specific temperature above the glass transition temperature (Tg) of the polymeric matrix for several hours. This results in melting of the surface layer on microspheres and therefore induces bonding of adjacent microspheres into a three-dimensional porous scaffold with excellent mechanical properties (4). Mechanical properties and porous structure of scaffolds are of great importance for bone repair. SMSs are built up by bonding of microspheres via surface fusion; therefore, sintering temperature and sintering time are generally recognized as crucial determinant factors. Higher sintering temperature and extended sintering time lead to stronger fusion of microspheres, smaller microsphere sizes, lower porosity and also smaller porous diameters, which give rise to a scaffold with better mechanical properties. On the contrary, lower sintering temperature and shorter sintering time induce weaker coalescent among the adjacent microspheres, which results in lower capability of SMSs to resist outside forces. Hence, optimization of sintering conditions would endow the scaffold with excellent characteristics for bony reconstruction, namely, desirable porosity and mechanical strength. Using a solvent to induce bonding of microspheres is another effective route to fabricate SMSs (5). Different solvents, such as methylene chloride and acetone (5,6), have been used to stick the separated microspheres into SMSs (Fig. 1). In comparison with heat sintering, solvent sintering provides milder means for manufacturing SMSs and thus Authors Xuetao Shi and Kai Su contributed equally to this manuscript. X. Shi : K. Su : R. R. Varshney :D.-A. Wang (*) Division of BioEngineering, School of Chemical & Biomedical Engineering Nanyang Technological University 70 Nanyang Drive, N1.3-B2-13 Singapore 637457 e-mail: DAWang@ntu.edu.sg

Transplantable delivery systems for in situ controlled release of bisphosphonate in orthopedic therapy

Importance of the field: Bisphosphonates (BPs), structurally similar to pyrophosphates and functionally superior in restraining osteoclast-induced bone resorption, have been widely used as clinical drugs in the treatment of osteoporosis, bone voids and associated inflammation. However, owing to their high aqueous solubility and the consequently high rate of loss during oral administration, the loading and targeting of BPs pose major challenges in practice. Alternative delivery routes such as nasal, subcutaneous/intramuscular injection have contributed little to improving the bioavailiability and efficacy of BPs. To improve and optimize the delivery efficiency and efficacy of BPs, numerous strategies have been developed and adopted. Studies on controlled release of BPs provide important information on the fabrication of BP delivery systems for in situ treatment. As BPs play an important therapeutic role in osteoporosis and similar diseases, it has become essential and vital to survey various reported fabrication methodologies of these systems and the consequential orthopedic treatments so as to keep abreast with advances in their clinical use. Areas covered in this review: Transplantable delivery systems for controlled release of BP are reviewed from literature published since 2000. The fabrication pathways and the release of BPs from various material systems are discussed in case studies. Recent progress in CaP models based on the strong and specific chelation between BPs and calcium phosphate crystals is highlighted. What the reader will gain: This review offers an outline of the advances in BP controlled release and delivery systems for orthopedic therapy. Take home message: Understanding the cutting-edge BP controlled release and delivery systems for in situ treatment is key to the successful design of a more promising and perfect delivery system for orthopedic therapy. Moreover, developing such delivery systems incorporating the numerous advantages of BPs and controlled release environment requires substantially more flexible models to control better the fate of BP drugs.