Blanka Sharma

Engineering structurally organized cartilage and bone tissues

The field of tissue engineering promises to deliver biological substitutes to repair or replace tissues in the body that have been injured or diseased. The clinical demand for musculoskeletal tissues is particularly high, especially for cartilage and bone defects. Although they are generally considered biologically simple structures, musculoskeletal tissues consist of highly organized three-dimensional networks of cells and matrix, giving rise to tissue structures with remarkable mechanical properties. Although the field of cartilage and bone tissue engineering has progressed significantly in recent years, the development of structurally ordered tissues has not been accomplished. More strategies are needed to ensure that the appropriate cell and matrix organization is being achieved in the engineered tissues. This review emphasizes how different cell types and scaffold designs can be used to modulate tissue properties and engineer more complex tissue structures, with emphasis on cartilage and bone tissues.

Experimental Model for Cartilage Tissue Engineering to Regenerate the Zonal Organization of Articular Cartilage

OBJECTIVE Regeneration of the zonal organization of articular cartilage may be an important advancement for cartilage tissue engineering. The first goal of this study was to validate our surgical technique as a method to selectively isolate chondrocytes from different zones of bovine articular cartilage. The second goal was to confirm that chondrocytes from different zones would have different proliferative and metabolic activities in two-dimensional (2-D) and 3-D cultures. Finally, to regenerate the zonal organization, we sought to make multi-layered constructs by encapsulating chondrocytes from different zones of articular cartilage. DESIGN Cartilage slices were removed from three (upper, middle, and lower) zones of articular cartilage of young bovine legs. Histology and biochemical composition of the cartilage slices were analyzed to confirm that they had been obtained from the proper zone. Growth kinetics and gene expression in monolayer culture and matrix formation in photopolymerizing hydrogels were evaluated. Multi-layered photopolymerizing hydrogels were constructed with chondrocytes from each zone of native cartilage encapsulated. Cell viability and maintenance of the cells in the respective layer were evaluated using the Live/Dead Viability kit and cell tracking protocols, respectively. After 3 weeks, the multi-layered constructs were harvested for histologic examination including immunohistochemistry for type II collagen. RESULTS Analysis of histology and biochemical composition confirmed that the cartilage slices had been obtained from the specific zone. Chondrocytes from different zones differed in growth kinetics and gene expression in monolayer and in matrix synthesis in 3-D culture. Cells encapsulated in each of the three layers of the hydrogel remained viable and remained in the respective layer in which they were encapsulated. After 3-week culture, each zone of multi-layered constructs had similar histologic findings to that of native articular cartilage. CONCLUSION We present this as an experimental model to regenerate zonal organization of articular cartilage by encapsulating chondrocytes from different layers in multi-layered photopolymerizing gels.

Adult stem cell driven genesis of human-shaped articular condyle.

Uniform design of synovial articulations across mammalian species is challenged by their common susceptibility to joint degeneration. The present study was designed to investigate the possibility of creating human-shaped articular condyles by rat bone marrow-derived mesenchymal stem cells (MSCs) encapsulated in a biocompatible poly(ethylene glycol)-based hydrogel. Rat MSCs were harvested, expanded in culture, and treated with either chondrogenic or osteogenic supplements. Rat MSC-derived chondrogenic and osteogenic cells were loaded in hydrogel suspensions in two stratified and yet integrated hydrogel layers that were sequentially photopolymerized in a human condylar mold. Harvested articular condyles from 4-week in vivo implantation demonstrated stratified layers of chondrogenesis and osteogenesis. Parallel in vitro experiments using goat and rat MSCs corroborated in vivo data by demonstrating the expression of chondrogenic and osteogenic markers by biochemical and mRNA analyses. Ex vivo incubated goat MSC-derived chondral constructs contained cartilage-related glycosaminoglycans and collagen. By contrast, goat MSC-derived osteogenic constructs expressed alkaline phosphatase and osteonectin genes, and showed escalating calcium content over time. Rat MSC-derived osteogenic constructs were stiffer than rat MSC-derived chondrogenic constructs upon nanoindentation with atomic force microscopy. These findings may serve as a primitive proof of concept for ultimate tissue-engineered replacement of degenerated articular condyles via a single population of adult mesenchymal stem cells.

Human Cartilage Repair with a Photoreactive Adhesive-Hydrogel Composite

A photoactive hydrogel is used in combination with microfracture to heal cartilage defects in patients. Let There Be Light Light has long been a favorite tool in medicine, finding utility in everything from skin conditions to depression to imaging. Now, Sharma and colleagues have shown that light can be used for biomaterials. Shining light on a hydrogel mixture causes it to polymerize within a defect, thus promoting tissue growth and repairing cartilage in patients. The biomaterial was designed to fill irregular wounds, such as articular cartilage defects. A biological adhesive was applied to the defect, followed by filling with a poly(ethylene glycol) (PEG)–based hydrogel solution. Then, light was applied to polymerize the material to form a solid implant. The hydrogel-adhesive was tested in a large-animal model to see how it worked in combination with the standard procedure for cartilage repair, called microfracture. The surgeons noted that the animals that received the biomaterial along with microfracture had a greater defect fill that was stronger and had more heterogeneous components (cells, proteins, etc.). The authors then moved to testing in people. Fifteen patients with symptomatic cartilage defects were treated with the adhesive-hydrogel after microfracture, whereas three patients were treated with microfracture only. No major adverse events were noted in 6 months after surgery. Similar to the animal studies, the photoactive biomaterial allowed for a greater filling of repair tissue in the defect compared with the control group, with material properties similar to adjacent, healthy cartilage. In addition, hydrogel-treated patients reported a decrease in overall pain severity and frequency over time. Although further clinical testing is needed to compare long-term outcomes in more patients, this light-mediated biomaterial therapy promises to be a versatile and safe way to enhance cartilage repair. Surgical options for cartilage resurfacing may be significantly improved by advances and application of biomaterials that direct tissue repair. A poly(ethylene glycol) diacrylate (PEGDA) hydrogel was designed to support cartilage matrix production, with easy surgical application. A model in vitro system demonstrated deposition of cartilage-specific extracellular matrix in the hydrogel biomaterial and stimulation of adjacent cartilage tissue development by mesenchymal stem cells. For translation to the joint environment, a chondroitin sulfate adhesive was applied to covalently bond and adhere the hydrogel to cartilage and bone tissue in articular defects. After preclinical testing in a caprine model, a pilot clinical study was initiated where the biomaterials system was combined with standard microfracture surgery in 15 patients with focal cartilage defects on the medial femoral condyle. Control patients were treated with microfracture alone. Magnetic resonance imaging showed that treated patients achieved significantly higher levels of tissue fill compared to controls. Magnetic resonance spin-spin relaxation times (T2) showed decreasing water content and increased tissue organization over time. Treated patients had less pain compared with controls, whereas knee function [International Knee Documentation Committee (IKDC)] scores increased to similar levels between the groups over the 6 months evaluated. No major adverse events were observed over the study period. With further clinical testing, this practical biomaterials strategy has the potential to improve the treatment of articular cartilage defects.

In vivo chondrogenesis of mesenchymal stem cells in a photopolymerized hydrogel.

Background: Surgical options for cartilage reconstruction can be significantly improved through advances in cartilage tissue engineering, whereby functional tissue replacements are created by growing cells on polymer scaffolds. The objective of this study was to use a photopolymerizable hydrogel to implant bone marrow–derived mesenchymal stem cells subcutaneously in a minimally invasive manner and promote cartilage tissue formation by the cells in vivo. Methods: Athymic nude mice were injected subcutaneously with polymer solutions of poly(ethylene) oxide diacrylate containing mesenchymal stem cells and placed under a UVA lamp to transdermally photopolymerize (solidify) the injected liquid. Experimental groups included polymer solutions with hyaluronic acid (HA), transforming growth factor (TGF)-β3, or both. After 3 weeks of implantation, cartilage formation was evaluated by gene expression analysis and histologic techniques. Results: Hyaluronic acid increased the viscosity of the polymer solutions, which helped maintain the injections at the desired site during photopolymerization. Mesenchymal stem cells in hydrogels containing both HA and TGF-β3 produced the highest quality cartilage, based on expression of the cartilage-specific genes and production of proteoglycan and collagen II. When used independently, TGF-β3 and HA alone induced cartilage-specific gene expression and collagen type II production; however, TGF-β3 was essential for proteoglycan production. HA enhanced proteoglycan production when combined with TGF-β3 and reduced expression and production of collagen I. Conclusions: This study is the first to demonstrate the minimally invasive implantation and subsequent chondrogenic differentiation of mesenchymal stem cells in the subcutaneous environment. This lays the foundation for further optimization of a novel and practical technology for cartilage reconstruction.

RESPONSE OF ZONAL CHONDROCYTES TO EXTRACELLULAR MATRIX-HYDROGELS

We investigated the biological response of chondrocytes isolated from different zones of articular cartilage and their cellular behaviors in poly (ethylene glycol)‐based (PEG) hydrogels containing exogenous type I collagen, hyaluronic acid (HA), or chondroitin sulfate (CS). The cellular morphology was strongly dependent on the extracellular matrix component of hydrogels. Additionally, the exogenous extracellular microenvironment affected matrix production and cartilage specific gene expression of chondrocytes from different zones. CS‐based hydrogels showed the strongest response in terms of gene expression and matrix accumulation for both superficial and deep zone chondrocytes, but HA and type I collagen‐based hydrogels demonstrated zonal‐dependent cellular responses.

Characterization of human mesenchymal stem cell-engineered cartilage: analysis of its ultrastructure, cell density and chondrocyte phenotype compared to native adult and fetal cartilage.

The production of engineered cartilage from mesenchymal stem cells is a rapidly developing field. Potential applications include the treatment of degenerative joint disease as well as the treatment of traumatic and surgical bone injury. Prior to clinical application, however, further characterization of the morphology, ultrastructure, biocompatibility, and performance of the engineered tissue is warranted. To achieve this, human mesenchymal stem cells (hMSCs) were grown in vitro in pellet culture for 3 weeks in chondrogenic medium conditions. The resultant engineered cartilage was compared to native adult and fetal tissue. Routine histology, special stains, and ultrastructural and quantitative histomorphometric analyses were performed. The engineered tissue demonstrated a similar chondrocyte phenotype, collagen fibril appearance, and matrix distribution when compared to native cartilage. By histomorphometric analysis, the cell density of the engineered cartilage was between that of native fetal and adult cartilage. The cell-to-matrix ratio and cellular area fraction of engineered cartilage samples was significantly greater than in adult samples, but indistinguishable from fetal cartilage samples, supporting the hypothesis that hMSC-engineered cartilage regeneration may mimic fetal cartilage development.

Progress in orthopedic biomaterials and drug delivery

Musculoskeletal conditions, including arthritis, back pain, and skeletal injuries/trauma, affect 50 % of the adult population in the U.S.A., and account for the second greatest cause of disability worldwide (www.bjdonline.org). The aggregate total annual cost of musculoskeletal diseases was

Two-Year Follow-Up and Remodeling Kinetics of ChonDux Hydrogel for Full-Thickness Cartilage Defect Repair in the Knee

796 billion in 2011. While most musculoskeletal injuries and diseases have lowmortality, they have a tremendous impact of quality of life and morbidity. Biomaterials have a long history in orthopedics—metal and plastic implants have been used over the last 60 years to treat end-stage joint degeneration and skeletal tissue loss, and alleviate the resulting pain and disability. First-generation orthopedic implants were designed to be biologically inert and selected primarily for their mechanical properties. However, they do not replicate the function of normal tissue and have a limited life span in the body, which is especially problematic in younger patients. Recent decades have focused on the development of biomaterials that are biodegradable and incorporate tissue-specific physicochemical cues to guide cell processes and tissue morphogenesis, with the goal of restoring natural tissue structure and function. These advances in biomaterials provide critical enabling technologies for the delivery of stem cells, gene therapies, and new biologics in the orthopedic field. This special issue highlights the recent advances and future directions in orthopedic biomaterials and drug delivery systems, and showcases emerging pharmaceutical and regenerative approaches to treat injuries, diseases, and disorders of the musculoskeletal system. Non-union bone fractures are a significant clinical challenge, and bone tissue engineering could have a key impact in improving current clinical options. Telvin et al. review the application of stem cells from various sources towards bone tissue engineering. The ability of transplanted stem cells to survive, continue to differentiate, and form bone tissue is key to making stem cell-mediated bone tissue regeneration approaches successful. The research article by Wen et al. precisely address this challenge. Their study compares the in vivo bone tissue forming ability of human bone marrow derived stem cells and human embryonic stem cells using osteoinductive mineralized materials as a scaffold. The article by Nyberg et al. reviews progress and challenges in achieving precise temporal and spatial control of growth factor presentation for functional bone tissue engineering ex vivo and in vivo. Ultimately, successful bone regeneration requires integration between the repair and native bone tissue. Botchway and colleagues address this critical issue, by modifying the surface of a bone allograft with FTY720, a synthetic analog of sphingosine 1-phosphate, to promote endogenous bone formation and osseous integration at the interface. Another key hurdle for the success of bone tissue engineering is vascularization in vivo. The review by Garcia and Garcia summarizes the current strategies and new developments targeted at vascularization of engineered bone, including growth factor and cell delivery, gene therapy, biomaterials, and combination approaches. The article by Tonello et al. demonstrates the potential of human placental matrix for promoting angiogenesis and its controlled release from biodegradable microparticles. In addition to bone, this special issue contains articles that highlight advances in cartilage tissue regeneration and osteoarthritis (OA) therapies. Osteoarthritis is the leading cause of * Blanka Sharma blanka.sharma@bme.ufl.edu

Oxidative Stress and Biomaterials: The Inflammatory Link

Objective To determine performance and repair kinetics of the ChonDux hydrogel scaffold for treating focal articular cartilage defects in the knee over 24 months. Design This assessor-blinded trial evaluates ChonDux hydrogel scaffold implantation in combination with microfracture in 18 patients across 6 sites. Male and female patients 18 to 65 years of age with full-thickness femoral condyle defects 2 to 4 cm2 in area were enrolled. Eligible patients received ChonDux treatment followed by rehabilitation. Defect volume fill was evaluated after 3, 6 (primary outcome), 12, 18, and 24 months by assessor blinded magnetic resonance imaging (MRI) analysis. Secondary outcomes were T2-weighted MRI relaxation time and patient surveys via visual analogue scale (VAS) pain and International Knee Documentation Committee (IKDC) knee function scoring. Results ChonDux maintained durable tissue restoration over 24 months with final defect percent fill of 94.2% ± 16.3% and no significant loss of fill volume at any time points. Tissues treated with ChonDux maintained T2 relaxation times similar to uninjured cartilage between 12 and 24 months. VAS pain scoring decreased between 1 and 6 weeks, and IKDC knee function scores improved by approximately 30.1 with ChonDux over 24 months. Conclusion ChonDux treatment is a safe adjunct to microfracture therapy and promotes stable restoration of full thickness articular cartilage defects for at least 24 months.