Charles W. Archer

Chondrogenesis of mesenchymal stem cells for cartilage tissue engineering

Despite its remarkable ability to resist mechanical loading, articular cartilage is not capable of mounting a useful reparative reaction in response to damage caused by trauma or disease. As a result numerous surgical and medical approaches have been developed to aid the healing of articular cartilage. Despite the success of surgical techniques such as microfracture, recently attentions have been turned to cell based therapies such as autologous chondrocyte implantation (ACI). ACI has produced encouraging results, however better results may be achievable through an evolution of this surgical approach. Since the first generation of ACI techniques changes have been made in the technique e.g. the introduction of collagen membranes instead of periosteal flaps, and more recently the use of collagen scaffolds for cellular delivery. The procedure has also moved on from being performed as an open operation and can now be performed arthroscopically. Despite these advances the procedure still uses chondrocytes harvested from the joint being repaired. These cells are vulnerable to dedifferentiation during the required in vitro expansion, and as a result may not be capable of producing repair tissue once implanted back into the joint. Mesenchymal stem cells (MSCs) may provide a dedifferentiation resistant alternative to chondrocytes. MSCs would also allow for the use of one arthroscopic operation on the affected joint, as opposed to the two operations that are currently required for ACI.

Evaluation of articular cartilage progenitor cells for the repair of articular defects in an equine model

BACKGROUND We sought to determine the effectiveness of chondroprogenitor cells derived from autologous and allogenic articular cartilage for the repair of cartilage defects in an equine model. METHODS Cartilage defects (15 mm) were created on the medial trochlear ridge of the femur. The following experimental treatments were compared with empty-defect controls: fibrin only, autologous chondroprogenitor cells plus fibrin, and allogenic chondroprogenitor cells plus fibrin (n = 4 or 12 per treatment). Horses underwent strenuous exercise throughout the twelve-month study, and evaluations included lameness (pain) and arthroscopic, radiographic, gross, histologic, and immunohistochemical analyses. RESULTS Arthroscopy and microscopy indicated that defects in the autologous cell group had significantly better repair tissue compared with defects in the fibrin-only and control groups. Repair tissue quality in the allogenic cell group was not superior to that in the fibrin-only group with the exception of the percentage of type-II collagen, which was greater. Radiographic changes in the allogenic cell group were poorer on average than those in the autologous cell group. Autologous cells significantly reduced central osteophyte formation compared with fibrin alone. CONCLUSIONS On the basis of the arthroscopic, radiographic, and histologic scores, autologous cells in fibrin yielded better results than the other treatments; allogenic cells cannot be recommended at this time.

Evidence of a Viable Pool of Stem Cells within Human Osteoarthritic Cartilage

Objectives Osteoarthritis (OA) is a debilitating disease affecting more than 4 million people in the United Kingdom. Despite its prevalence, there is no successful cell-based therapy currently used to treat patients whose cartilage is deemed irrecoverable. The present study aimed to isolate stem cells from tibial plateaux cartilage obtained from patients who underwent total knee replacements for OA and investigate their stem cell characteristics. Design Clonally derived cell lines were selected using a differential adhesion assay to fibronectin and expanded in monolayer culture. Colony forming efficiencies and growth kinetics were investigated. The potential for tri-lineage differentiation into chondrogenic, osteogenic, and adipogenic phenotypes were analyzed using histological stains, immunocytochemistry, and reverse transcriptase polymerase chain reaction. Results Colony forming cells were successfully isolated from osteoarthritic cartilage and extensively expanded in monolayer culture. Colony forming efficiencies were consistently below 0.1%. Clonal cell lines were expanded beyond 40 population doublings but disparities were observed in the number of population doublings per day. Clonally derived cell lines also demonstrated in vitro multilineage potential via successful differentiation into chondrogenic, osteogenic, and adipogenic lineages. However, variation in the degree of differentiation was observed between these clonal cell lines. Conclusions A viable pool of cells with stem cell characteristics have been identified within human osteoarthritic cartilage. Variation in the degree of differentiation suggests the possibility of further subpopulations of cells. The identification of this stem cell population highlights the reparative potential of these cells in osteoarthritic cartilage, which could be further exploited to aid the field of regenerative medicine.

Human Articular Cartilage Progenitor Cells Are Responsive to Mechanical Stimulation and Adenoviral-Mediated Overexpression of Bone-Morphogenetic Protein 2

Articular cartilage progenitor cells (ACPCs) represent a new and potentially powerful alternative cell source to commonly used cell sources for cartilage repair, such as chondrocytes and bone-marrow derived mesenchymal stem cells (MSCs). This is particularly due to the apparent resistance of ACPCs to hypertrophy. The current study opted to investigate whether human ACPCs (hACPCs) are responsive towards mechanical stimulation and/or adenoviral-mediated overexpression of bone morphogenetic protein 2 (BMP-2). hACPCs were cultured in fibrin-polyurethane composite scaffolds. Cells were cultured in a defined chondro-permissive medium, lacking exogenous growth factors. Constructs were cultured, for 7 or 28 days, under free-swelling conditions or with the application of complex mechanical stimulation, using a custom built bioreactor that is able to generate joint-like movements. Outcome parameters were quantification of BMP-2 and transforming growth factor beta 1 (TGF-β1) concentration within the cell culture medium, biochemical and gene expression analyses, histology and immunohistochemistry. The application of mechanical stimulation alone resulted in the initiation of chondrogenesis, demonstrating the cells are mechanoresponsive. This was evidenced by increased GAG production, lack of expression of hypertrophic markers and a promising gene expression profile (significant up-regulation of cartilaginous marker genes, specifically collagen type II, accompanied by no increase in the hypertrophic marker collagen type X or the osteogenic marker alkaline phosphatase). To further investigate the resistance of ACPCs to hypertrophy, overexpression of a factor associated with hypertrophic differentiation, BMP-2, was investigated. A novel, three-dimensional, transduction protocol was used to transduce cells with an adenovirus coding for BMP-2. Over-expression of BMP-2, independent of load, led to an increase in markers associated with hypertropy. Taken together ACPCs represent a potential alterative cell source for cartilage tissue engineering applications.

Asymmetrical seeding of MSCs into fibrin–poly(ester-urethane) scaffolds and its effect on mechanically induced chondrogenesis

Mesenchymal stem cells (MSCs) are currently being investigated as candidate cells for regenerative medicine approaches for the repair of damaged articular cartilage. For these cells to be used clinically, it is important to understand how they will react to the complex loading environment of a joint in vivo. In addition to investigating alternative cell sources, it is also important for the structure of tissue‐engineered constructs and the organization of cells within them to be developed and, if possible, improved. A custom built bioreactor was used to expose human MSCs to a combination of shear and compression loading. The MSCs were either evenly distributed throughout fibrin‐poly(ester‐urethane) scaffolds or asymmetrically seeded with a small proportion seeded on the surface of the scaffold. The effect of cell distribution on the production and deposition of cartilage‐like matrix in response to mechanical load mimicking in vivo joint loading was then investigated. The results show that asymmetrically seeding the scaffold led to markedly improved tissue development based on histologically detectable matrix deposition. Consideration of cell location, therefore, is an important aspect in the development of regenerative medicine approaches for cartilage repair. This is particularly relevant when considering the natural biomechanical environment of the joint in vivo and patient rehabilitation protocols.

Regenerative medicine and biomaterials for the repair of connective tissues

Structure and regenerative capacity of synovial joint tissues The myofibroblast in connective tissue repair and regeneration. Part 1 Cartilage repair and regeneration: Structure of articular cartilage Measuring the biomechanical properties of cartilage cells Understanding tissue response to cartilage injury Understanding osteoarthritis and other cartilage diseases Using animal models of cartilage repair to screen new clinical techniques Cartilage tissue repair: autologous osteochondral mosaicplasty Cartilage tissue repair: autologous chondrocyte implantation Cell sheet technologies for cartilage repair Cell therapies for articular cartilage repair: chondrocytes and mesenchymal stem cells Scaffolds for musculoskeletal tissue engineering Outcome measures of articular cartilage repair. Part 2 Repair of tendons and ligaments: The structure of tendons and ligaments Tendon biomechanics Tendon injury and repair mechanisms Tissue engineering for ligament and tendon repair Cell-based therapies for the repair and regeneration of tendons and ligaments Scaffolds for tendon and ligament tissue engineering.

Tissue-Engineered Solutions in Plastic and Reconstructive Surgery: Principles and Practice

Recent advances in microsurgery, imaging, and transplantation have led to significant refinements in autologous reconstructive options; however, the morbidity of donor sites remains. This would be eliminated by successful clinical translation of tissue-engineered solutions into surgical practice. Plastic surgeons are uniquely placed to be intrinsically involved in the research and development of laboratory engineered tissues and their subsequent use. In this article, we present an overview of the field of tissue engineering, with the practicing plastic surgeon in mind. The Medical Research Council states that regenerative medicine and tissue engineering “holds the promise of revolutionizing patient care in the twenty-first century.” The UK government highlighted regenerative medicine as one of the key eight great technologies in their industrial strategy worthy of significant investment. The long-term aim of successful biomanufacture to repair composite defects depends on interdisciplinary collaboration between cell biologists, material scientists, engineers, and associated medical specialties; however currently, there is a current lack of coordination in the field as a whole. Barriers to translation are deep rooted at the basic science level, manifested by a lack of consensus on the ideal cell source, scaffold, molecular cues, and environment and manufacturing strategy. There is also insufficient understanding of the long-term safety and durability of tissue-engineered constructs. This review aims to highlight that individualized approaches to the field are not adequate, and research collaboratives will be essential to bring together differing areas of expertise to expedite future clinical translation. The use of tissue engineering in reconstructive surgery would result in a paradigm shift but it is important to maintain realistic expectations. It is generally accepted that it takes 20–30 years from the start of basic science research to clinical utility, demonstrated by contemporary treatments such as bone marrow transplantation. Although great advances have been made in the tissue engineering field, we highlight the barriers that need to be overcome before we see the routine use of tissue-engineered solutions.

Combining regenerative medicine strategies to provide durable reconstructive options: auricular cartilage tissue engineering

Recent advances in regenerative medicine place us in a unique position to improve the quality of engineered tissue. We use auricular cartilage as an exemplar to illustrate how the use of tissue-specific adult stem cells, assembly through additive manufacturing and improved understanding of postnatal tissue maturation will allow us to more accurately replicate native tissue anisotropy. This review highlights the limitations of autologous auricular reconstruction, including donor site morbidity, technical considerations and long-term complications. Current tissue-engineered auricular constructs implanted into immune-competent animal models have been observed to undergo inflammation, fibrosis, foreign body reaction, calcification and degradation. Combining biomimetic regenerative medicine strategies will allow us to improve tissue-engineered auricular cartilage with respect to biochemical composition and functionality, as well as microstructural organization and overall shape. Creating functional and durable tissue has the potential to shift the paradigm in reconstructive surgery by obviating the need for donor sites.

Phenotype and Viability of MLO-Y4 Cells Is Maintained by TGFβ3 in a Serum-Dependent Manner within a 3D-Co-Culture with MG-63 Cells

The osteocyte network inside the bone matrix is of functional importance and osteocyte cell death is a characteristic feature of pathological bone diseases. Osteocytes have emerged as key regulators of bone tissue maintenance, yet maintaining their phenotype during in vitro culture remains challenging. A 3D co-culture system for osteocytes with osteoblasts was recently presented, enabling the determination of more physiological effects of growth factors on cells in vitro. MLO-Y4 cells were embedded within a type I collagen gel and cultured in the presence of surface MG-63 cells. Co-culture was performed in the presence or absence of TGFβ3. Gene expression by quantitative PCR, protein expression by fluorescent immunohistochemistry and cell viability tests were performed. The 3D co-culture induced cell differentiation of MG-63 cells seen by increased type I collagen and osteocalcin mRNA expression. TGFβ3 maintained osteocyte differentiation of MLO-Y4 cells during co-culture as determined by stable E11 and osteocalcin mRNA expression till day 4. Interestingly, most of the effects of TGFβ3 on co-cultured cells were serum-dependent. Also, TGFβ3 reduced cell death of 3D co-cultured MLO-Y4 cells in a serum-dependent manner. This study shows that 3D co-culture upregulates differentiation of MG-63 cells to a more mature osteoblast-like phenotype; while the addition of TGFβ3 maintained the characteristic MLO-Y4 osteocyte-like phenotype and viability in a serum-dependent manner.

Bovine articular chondroprogenitors: tools for cartilage tissue engineering [Abstract]

Introduction The use of stem/progenitor cells whose progeny have the capacity to form good quality cartilage matrix has been targeted as a therapeutic goal for the treatment of large defects of articular cartilage. Previously we reported the isolation and characterisation of a progenitor cell population that resides in the surface layer of neonatal bovine articular cartilage. Here we show that chondroprogenitors maintain sox9 expression, telomere length and telomerase activity following clonal expansion. Materials and Methods Telomere length and telomerase activity of samples was assayed using Southern blotting and TRAP assay, respectively. Pellet culture utilised 1 · 106 cells grown in high-density culture in chondrogenic medium for 4 weeks. Results Cloned chondroprogenitors exhibited exponential growth for the first 20 population doublings (PD), then slower linear growth with evidence of replicative senescence at later passages. Mean telomere lengths of exponentially growing chondroprogenitors were significantly longer than dedifferentiated chondrocytes that had undergone a similar number of PD (P < 0.05). Chondroprogenitors also had 2.6-fold greater telomerase activity and had similar sox9 and had reduced Notch-1 gene expression levels when compared to dedifferentiated chondrocytes. Chondroprogenitors were induced to differentiate into cartilage in 3D pellet cultures, immunological investigation of sox9, Notch- 1, aggrecan and PCNA expression showed evidence of coordinated growth and differentiation within the cartilage pellet. Discussion Clonal chondroprogenitors are a useful tool to analyse progenitor cell growth and differentiation chracteristics. Whether chondroprogenitor cells from immature articular cartilage differ from those derived from more mature tissue may shed light on the variable nature of cell-based cartilage repair therapies.