Tissue engineering applications: cartilage lesions repair by the use of autologous chondrocytes

Promising new therapies based on tissue engineering have been recently developed for cartilage repair. The association of biomaterials with autologous chondrocytes expanded in vitro can represent a useful tool to regenerate this tissue. The scaffolds utilised in such therapeutical applications should provide a pre-formed three-dimensional shape, prevent cells from floating out of the defect, have sufficient mechanical strength, facilitate uniform spread of cells and stimulate the phenotype of transplanted cells. Hyaff-11 is a hyaluronic-acid based biodegradable polymer, that has been shown to provide successful cell carrier for tissue-engineered repair. From our findings we can state that human chondrocytes seeded on Hyaff-11 are able to maintain in vitro the characteristic of differentiated cells, expressing and producing collagen type II and aggrecan which are the main markers of cartilage phenotype, down-regulating collagen type I. Moreover, it seems to be a useful scaffold for cartilage repair both in animal models and clinical trials in humans, favouring the formation of a hyaline-like tissue. In the light of these data, we can hypothesise, for the future, the use of autologous chondrocyte transplantation together with gene therapy as a treatment for rheumatic diseases such as osteoarthritis.     

Premature induction of hypertrophy during in vitro chondrogenesis of human mesenchymal stem cells correlates with calcification and vascular invasion after ectopic transplantation in SCID mice

OBJECTIVE: Functional suitability and phenotypic stability of ectopic transplants are crucial factors in the clinical application of mesenchymal stem cells (MSCs) for articular cartilage repair, and might require a stringent control of chondrogenic differentiation. This study evaluated whether human bone marrow-derived MSCs adopt natural differentiation stages during induction of chondrogenesis in vitro, and whether they can form ectopic stable cartilage that is resistant to vascular invasion and calcification in vivo. METHODS: During in vitro chondrogenesis of MSCs, the expression of 44 cartilage-, stem cell-, and bone-related genes and the deposition of aggrecan and types II and X collagen were determined. Similarly treated, expanded articular chondrocytes served as controls. MSC pellets were allowed to differentiate in chondrogenic medium for 3-7 weeks, after which the chondrocytes were implanted subcutaneously into SCID mice; after 4 weeks in vivo, samples were evaluated by histology. RESULTS: The 3-stage chondrogenic differentiation cascade initiated in MSCs was primarily characterized by sequential up-regulation of common cartilage genes. Premature induction of hypertrophy-related molecules (type X collagen and matrix metalloproteinase 13) occurred before production of type II collagen and was followed by up-regulation of alkaline phosphatase activity. In contrast, hypertrophy-associated genes were not induced in chondrocyte controls. Whereas control chondrocyte pellets resisted calcification and vascular invasion in vivo, most MSC pellets mineralized, in spite of persisting proteoglycan and type II collagen content. CONCLUSION: An unnatural pathway of differentiation to chondrocyte-like cells was induced in MSCs by common in vitro protocols. MSC pellets transplanted to ectopic sites in SCID mice underwent alterations related to endochondral ossification rather than adopting a stable chondrogenic phenotype. Further studies are needed to evaluate whether a more stringent control of MSC differentiation to chondrocytes can be achieved during cartilage repair in a natural joint environment.     

Adenoviral-mediated transfer of TGF-beta1 but not IGF-1 induces chondrogenic differentiation of human mesenchymal stem cells in pellet cultures

OBJECTIVE: The objective of the present study was to investigate the potential of application of growth factor genes to induce chondrogenic differentiation of human-derived mesenchymal stem cells (MSCs). The growth factor genes evaluated in the present study were transforming growth factor 1 (TGF-beta1) and insulin-like growth factor 1 (IGF-1). METHODS: Human MSCs were transduced with the adenoviral vectors carrying either TGF-beta1 or IGF-1 (AdTGF-beta1 and AdIGF-1 respectively) or a combination of both growth factor genes at different multiplicities of infection (MOI) and were then made into pellets. Pellets were also made from nontransduced cells and maintained in culture medium supplemented with 10 ng/mL of TGF-beta1. At specified time points, histological analysis, cartilage matrix gene expression, and immunofluorescence were performed to determine the extent of chondrogenic differentiation. RESULTS: MSCs transduced with the AdTGF-beta1 demonstrated robust chondrogenic differentiation, while those made from AdIGF-1 did not. AdTGF-beta1 pellets demonstrated aggrecan gene expression as early as day 3 of pellet culture, while type II collagen gene expression was detected by day 10 of culture. The AdIGF-1, alone or in combination with TGF-beta1 pellets, did not show any type II collagen gene expression at any time point. By immunofluoresecence, type X collagen was distributed throughout the matrix in TGF-beta1 protein pellets while the growth factor gene pellets displayed scant staining. CONCLUSION: The results suggest that sustained administration of TGF-beta1 may be more effective in suppressing terminal differentiation than intermittent dosing and thus effective for cartilage repair.     

Enhancement of human platelet activation by the combination of low concentrations of collagen and rabbit anticardiolipin antibodies

Low concentrations of collagen and anticardiolipin antibodies (ACLA), which were raised in rabbits by immunization with cardiolipin (CL), co-operatively activated human gel-filtrated platelets (GFP). GFP activated by adding ACLA 5 min prior to collagen (ACLA + Col) showed strong responses in cytosolic Ca2+ mobilization and cell aggregation; the responses decreased after 1 min, however, when collagen was added prior to ACLA (Col + ACLA). Col + ACLA was 30% less effective than the ACLA + Col in: (1) the phosphorylation of pleckstrin and myosin light chain; and (2) the secretion of alpha- and dense granules. Indomethacin inhibited Ca2+ mobilization, pleckstrin phosphorylation and cell aggregation in platelets stimulated by ACLA + Col. The thromboxane B2 level in platelets induced by ACLA + Col was similar to that stimulated by low concentrations of collagen alone. ACLA + Col increased the activities of phospholipase C (PLC) as determined by formation of phosphatidic acid (PA), whereas indomethacin and adenosine 2',5'-diphosphate, an antagonist of the ADP P2Y1 receptor, inhibited PA formation. These results suggest that ACLA, thromboxane A2 derived from the collagen pathway and secreted ADP co-operatively augment PLC activity and lead to platelet aggregation.     

Thinking outside the liver: Induced pluripotent stem cells for hepatic applications

The discovery of induced pluripotent stem cells (iPSCs) unraveled a mystery in stem cell research, after identification of four re-programming factors for generating pluripotent stem cells without the need of embryos. This breakthrough in generating iPSCs from somatic cells has overcome the ethical issues and immune rejection involved in the use of human embryonic stem cells. Hence, iPSCs form a great potential source for developing disease models, drug toxicity screening and cell-based therapies. These cells have the potential to differentiate into desired cell types, including hepatocytes, under in vitro as well as under in vivo conditions given the proper microenvironment. iPSC-derived hepatocytes could be useful as an unlimited source, which can be utilized in disease modeling, drug toxicity testing and producing autologous cell therapies that would avoid immune rejection and enable correction of gene defects prior to cell transplantation. In this review, we discuss the induction methods, role of reprogramming factors, and characterization of iPSCs, along with hepatocyte differentiation from iPSCs and potential applications. Further, we discuss the location and detection of liver stem cells and their role in liver regeneration. Although tumor formation and genetic mutations are a cause of concern, iPSCs still form a promising source for clinical applications.     

In vivo commitment and functional tissue regeneration using human embryonic stem cell-derived mesenchymal cells

Development of clinically relevant regenerative medicine therapies using human embryonic stem cells (hESCs) requires production of a simple and readily expandable cell population that can be directed to form functional 3D tissue in an in vivo environment. We describe an efficient derivation method and characterization of mesenchymal stem cells (MSCs) from hESCs (hESCd-MSCs) that have multilineage differentiation potential and are capable of producing fat, cartilage, and bone in vitro. Furthermore, we highlight their in vivo survival and commitment to the chondrogenic lineage in a microenvironment comprising chondrocyte-secreted morphogenetic factors and hydrogels. Normal cartilage architecture was established in rat osteochondral defects after treatment with chondrogenically-committed hESCd-MSCs. In view of the limited available cell sources for tissue engineering applications, these embryonic-derived cells show significant potential in musculoskeletal tissue regeneration applications.     

Cartilage restoration in haemophilia: advanced therapies

Summary. Current treatment of joint cartilage lesions is based either on conventional techniques (bone marrow stimulation, osteochondral autograft or allograft transplantation) or on newly developed techniques (chondrocyte implantation and those based on cell therapy that use bioreactors, growth factors, mesenchymal stem cells [MSCs] and genetically modified cells). The aim of this article is to review the therapeutic strategies above mentioned and to determine whether the chondral damage seen in haemophilia could benefit from any of them. The different conventional techniques have shown similar results whereas autologous chondrocyte implantation, which is in common use at the present time, has not been shown to produce any conclusive results or to lead to the formation of hyaline cartilage. MSCs hold promise for the repair of joint cartilage given their differentiation capacity and the therapeutic effect. The use of bioreactors and growth factors, which stimulate cartilage formation, may optimize such strategies in the context of reimplantation of chondrocytes, differentiated MSCs and cartilage progenitor cells. The aim of cell therapy is restoration of function through the repair of damaged tissue or the stimulation of growth factor synthesis. Implantation of autologous chondrocytes or MSCs was up to now able to address only highly localized chondral lesions. Adequate control of the differentiation process as well as the use of growth factors and appropriate bioreactors could transform cell-based therapies into a more efficient and longer term treatment even for patients with haemophilia. Nevertheless, raising false expectations in these patients should be avoided. There are a number of approaches to cartilage restoration in haemophilic arthropathy, which are currently being explored for other joint related degenerative disorders. If it can be proven to be effective for the disorders in which clinical trials are ongoing and costs could be limited, it might be an useful palliative approach to haemophilic arthropathy. However, we still have a long way to go for use in haemophilic arthropathy.     

Methodological models for in vitro amplification and maintenance of human articular chondrocytes from elderly patients

Articular cartilage defects, an exceedingly common problem closely correlated with advancing age, is characterized by lack of spontaneous resolution because of the limited regenerative capacity of adult articular chondrocytes. Medical and surgical therapies yield unsatisfactory short-lasting results. Recently, cultured autologous chondrocytes have been proposed as a source to promote repair of deep cartilage defects. Despite encouraging preliminary results, this approach is not yet routinely applicable in clinical practice, but for young patients. One critical points is the isolation and ex vivo expansion of large enough number of differentiated articular chondrocytes. In general, human articular chondrocytes grown in monolayer cultures tend to undergo dedifferentiation. This reversible process produces morphological changes by which cells acquire fibroblast-like features, loosing typical functional characteristics, such as the ability to synthesize type II collagen. The aim of this study was to isolate human articular chondrocytes from elderly patients and to carefully characterize their morphological, proliferative, and differentiative features. Cells were morphologically analyzed by optic and transmission electron microscopy (TEM). Production of periodic acid-schiff (PAS)-positive cellular products and of type II collagen mRNA was monitored at different cellular passages. Typical chondrocytic characteristics were also studied in a suspension culture system with cells encapsulated in alginate-polylysine-alginate (APA) membranes. Results showed that human articular chondrocytes can be expanded in monolayers for several passages, and then microencapsulated, retaining their morphological and functional characteristics. The results obtained could contribute to optimize expansion and redifferentiation sequences for applying cartilage tissue engineering in the elderly patients.     

骨关节炎患者膝关节软骨细胞的离体培养及形态学特征观察

目的建立膝关节患者关节软骨细胞体外培养体系,为其相关研究奠定基础。方法取15例人工膝关节置换术患者的软骨组织,采用组织原代培养法获得膝关节软骨细胞,传代培养。倒置相差显微镜观察细胞形态,绘制生长曲线,苏木素伊红染色、甲苯胺蓝染色及Ⅰ型胶原、Ⅱ型胶原、X型胶原、MMP-1、MMP-13、TNF-α免疫荧光染色对细胞进行鉴定。结果原代培养的人软骨细胞大量呈梭形、短梭形,少部分呈多角形,传代3次后出现树突生长细胞。形态学、免疫荧光染色显示细胞培养3代以内可保持表型稳定,多数软骨细胞维持梭形、短梭形,核为圆形或椭圆形。苏木精伊红阳性染色为细胞核呈紫蓝色,软骨细胞基质呈红色,周围胞浆呈紫红色、阿利新蓝染色后,胞质和胞膜呈深蓝色、Ⅱ型胶原免疫荧光染色可见胞质和胞膜清晰绿色荧光,细胞核区域未见明显绿色荧光。结论成功建立了的软骨细胞分离、培养体系,且3代以内细胞生长良好。     

Self-assembling peptide hydrogel fosters chondrocyte extracellular matrix production and cell division: implications for cartilage tissue repair

Emerging medical technologies for effective and lasting repair of articular cartilage include delivery of cells or cell-seeded scaffolds to a defect site to initiate de novo tissue regeneration. Biocompatible scaffolds assist in providing a template for cell distribution and extracellular matrix (ECM) accumulation in a three-dimensional geometry. A major challenge in choosing an appropriate scaffold for cartilage repair is the identification of a material that can simultaneously stimulate high rates of cell division and high rates of cell synthesis of phenotypically specific ECM macromolecules until repair evolves into steady-state tissue maintenance. We have devised a self-assembling peptide hydrogel scaffold for cartilage repair and developed a method to encapsulate chondrocytes within the peptide hydrogel. During 4 weeks of culture in vitro, chondrocytes seeded within the peptide hydrogel retained their morphology and developed a cartilage-like ECM rich in proteoglycans and type II collagen, indicative of a stable chondrocyte phenotype. Time-dependent accumulation of this ECM was paralleled by increases in material stiffness, indicative of deposition of mechanically functional neo-tissue. Taken together, these results demonstrate the potential of a self-assembling peptide hydrogel as a scaffold for the synthesis and accumulation of a true cartilage-like ECM within a three-dimensional cell culture for cartilage tissue repair.     

Identification of mesenchymal progenitor cells in normal and osteoarthritic human articular cartilage

OBJECTIVE: To determine the presence of mesenchymal progenitor cells (MPCs) in human articular cartilage. METHODS: Primary cell cultures established from normal and osteoarthritic (OA) human knee articular cartilage were analyzed for the expression of CD105 and CD166, cell surface markers whose coexpression defines mesenchymal stem cells (MSCs) in bone marrow and perichondrium. The potential of cartilage cells to differentiate to adipogenic, osteogenic, and chondrogenic lineages was analyzed after immunomagnetic selection for CD105+/CD166+ cells and was compared with bone marrow-derived MSCs (BM-MSCs). RESULTS: Up to 95% of isolated cartilage cells were CD105+ and approximately 5% were CD166+. The mean +/- SEM percentage of CD105+/CD166+ cells in normal cartilage was 3.49 +/- 1.93%. Primary cell cultures from OA cartilage contained significantly increased numbers of CD105+/CD166+ cells. Confocal microscopy confirmed the coexpression of both markers in the majority of BM-MSCs and a subpopulation of cartilage cells. Differentiation to adipocytes occurred in cartilage-derived cell cultures, as indicated by characteristic cell morphology and oil red O staining of lipid vacuoles. Osteogenesis was observed in isolated CD105+/CD166+ cells as well as in primary chondrocytes cultured in the presence of osteogenic supplements. Purified cartilage-derived CD105+/CD166+ cells did not express markers of differentiated chondrocytes. However, the cells were capable of chondrocytic differentiation and formed cartilage tissue in micromass pellet cultures. CONCLUSION: These findings indicate that multipotential MPCs are present in adult human articular cartilage and that their frequency is increased in OA cartilage. This observation has implications for understanding the intrinsic repair capacity of articular cartilage and raises the possibility that these progenitor cells might be involved in the pathogenesis of arthritis.     

Small molecules and extrinsic factors promoting differentiation of stem cells into insulin-producing cells

In the modern world, type-2 diabetes mellitus has become a leading public healthcare problem, due to major risks of morbidity and mortality. Prevalence has increased significantly in recent decades. Treatment involves oral hypoglycemic agents or insulin replacement therapy. Development is ongoing for cell-based diabetes therapies using stem cells with the potential to differentiate into insulin-producing cells (IPCs): embryonic stem cells (ESCs), mesenchymal stem cells (MSCs), induced pluripotent stem cells (iPSCs), and stem cells from adult pancreas, liver, central nervous system, bone marrow and adipose tissue. Successful induction of iPSCs, however, depends on the quantity and quality of available stem cells and the development of adapted protocols determining the environment of extrinsic factors and involvement of small molecules. Validating such new cell therapies must be founded on this experimental rationale.     

Bone marrow transplantation restores epidermal basement membrane protein expression and rescues epidermolysis bullosa model mice

Attempts to treat congenital protein deficiencies using bone marrow-derived cells have been reported. These efforts have been based on the concepts of stem cell plasticity. However, it is considered more difficult to restore structural proteins than to restore secretory enzymes. This study aims to clarify whether bone marrow transplantation (BMT) treatment can rescue epidermolysis bullosa (EB) caused by defects in keratinocyte structural proteins. BMT treatment of adult collagen XVII (Col17) knockout mice induced donor-derived keratinocytes and Col17 expression associated with the recovery of hemidesmosomal structure and better skin manifestations, as well improving the survival rate. Both hematopoietic and mesenchymal stem cells have the potential to produce Col17 in the BMT treatment model. Furthermore, human cord blood CD34⁺ cells also differentiated into keratinocytes and expressed human skin component proteins in transplanted immunocompromised (NOD/SCID/γ_c^null) mice. The current conventional BMT techniques have significant potential as a systemic therapeutic approach for the treatment of human EB.     

Cartilage repair in a rat model of osteoarthritis through intraarticular transplantation of muscle‐derived stem cells expressing bone morphogenetic protein 4 and soluble flt‐1

Objective

The control of angiogenesis during chondrogenic differentiation is an important issue affecting the use of stem cells in cartilage repair, especially with regard to the persistence of regenerated cartilage. This study was undertaken to investigate the effect of vascular endothelial growth factor (VEGF) stimulation and the blocking of VEGF with its antagonist, soluble Flt‐1 (sFlt‐1), on the chondrogenesis of skeletal muscle‐derived stem cells (MDSCs) in a rat model of osteoarthritis (OA).

Methods

We investigated the effect of VEGF on cartilage repair in an immunodeficiency rat model of OA after intraarticular injection of murine MDSCs expressing bone morphogenetic protein 4 (BMP‐4) in combination with MDSCs expressing VEGF or sFlt‐1.

Results

In vivo, a combination of sFlt‐1– and BMP‐4–transduced MDSCs demonstrated better repair without osteophyte formation macroscopically and histologically following OA induction, when compared with the other groups. Higher differentiation/proliferation and lower levels of chondrocyte apoptosis were also observed in sFlt‐1– and BMP‐4–transduced MDSCs compared with a combination of VEGF‐ and BMP‐4–transduced MDSCs or with BMP‐4–transduced MDSCs alone. In vitro experiments with mixed pellet coculture of MDSCs and OA chondrocytes revealed that BMP‐4–transduced MDSCs produced the largest pellets, which had the highest gene expression of not only type II collagen and SOX9 but also type X collagen, suggesting formation of hypertrophic chondrocytes.

Conclusion

Our results demonstrate that MDSC‐based therapy involving sFlt‐1 and BMP‐4 repairs articular cartilage in OA mainly by having a beneficial effect on chondrogenesis by the donor and host cells as well as by preventing angiogenesis, which eventually prevents cartilage resorption, resulting in persistent cartilage regeneration and repair.

     

Hepatic precursors derived from murine embryonic stem cells contribute to regeneration of injured liver

We established an efficient system for differentiation, expansion and isolation of hepatic progenitor cells from mouse embryonic stem (ES) cells and evaluated their capacity to repopulate injured liver. Using mouse ES cells transfected with the green fluorescent protein (GFP) reporter gene regulated by albumin (ALB) enhancer/promoter, we found that a serum-free chemically defined medium supports formation of embryoid bodies (EBs) and differentiation of hepatic lineage cells in the absence of exogenous growth factors or feeder cell layers. The first GFP+ cells expressing ALB were detected in close proximity to "beating" myocytes after 7 days of EB cultures. GFP+ cells increased in number, acquired hepatocyte-like morphology and hepatocyte-specific markers (i.e., ALB, AAT, TO, and G6P), and by 28 days represented more than 30% of cells isolated from EB outgrowths. The FACS-purified GFP+ cells developed into functional hepatocytes without evidence of cell fusion and participated in the repairing of diseased liver when transplanted into MUP-uPA/SCID mice. The ES cell-derived hepatocytes were responsive to normal growth regulation and proliferated at the same rate as the host hepatocytes after an additional growth stimulus from CCl(4)-induced liver injury. The transplanted GFP+ cells also differentiated into biliary epithelial cells. In conclusion, a highly enriched population of committed hepatocyte precursors can be generated from ES cells in vitro for effective cell replacement therapy.     

Analysis of cartilage biomarkers in the early phases of canine experimental osteoarthritis

OBJECTIVE: To study 3 body fluids for changes in the levels of 5 biomarkers of cartilage metabolism during the early phases of experimental osteoarthritis (OA). METHODS: Twenty skeletally mature mixed-breed canines underwent unilateral surgical transection of the anterior cruciate ligament. Samples of joint fluid, serum, and urine were obtained preoperatively and just before necropsy (3 weeks or 12 weeks postoperatively). Biomarkers included 2 markers of cartilage matrix synthesis/turnover (aggrecan 846 epitope and C-propeptide of type II collagen) and 3 markers of cartilage degradation (keratan sulfate proteoglycan epitope, the collagenase-generated cleavage epitope of type II collagen [Col2-3/4C(long mono), or CIIC], and crosslinked peptides from the C-telopeptide domain of type II collagen [Col2CTx]). Significant changes in the levels of these biomarkers were determined by paired analyses. RESULTS: Joint pathology was more severe in the 12-week group compared with the 3-week group. In joint fluid, due to limited volume, only Col2-3/4C(long mono) and Col2CTx were measured. Significant elevations in the levels of both of these markers were observed in experimental joints in both the 3-week group and the 12-week group. In serum, the level of aggrecan 846 epitope was elevated at both 3 weeks and 12 weeks, the level of Col2-3/4C(long mono) was elevated at 12 weeks, and the level of Col2CTx was elevated at both 3 weeks and 12 weeks. In urine, the level of Col2-3/4C(long mono) was elevated at 12 weeks after surgery. CONCLUSION: Levels of biomarkers of intact aggrecan proteoglycan (aggrecan 846 epitope) and type II collagen degradation (Col2-3/4C(long mono) and Col2CTx) were elevated early after unilateral stifle joint injury, suggesting that these markers are sensitive and specific for early cartilage changes associated with isolated joint injury in this established model of experimental OA.