Greater Chondrogenic Potential Research Articles

A Proteomic Approach to Determine Stem Cell Skeletal Differentiation Signature on Additive Manufactured Scaffolds

Understanding how porous biomaterials interact with cells at their surface and how they either promote or inhibit cellular processes has presented several challenges. Additive manufacturing enables the fabrication of scaffolds with distinct compositions and designs for different tissue engineering applications. To evaluate the in vitro performance of multiple printed materials, biochemical assays can be limiting in providing valuable insight and key information to select the best tissue destination. Omics technologies like proteomics are crucial for studying important cellular events and gathering valuable information about cellular processes and mechanisms. However, only few studies focus on proteomics to decipher cell–material interactions and cell differentiation on additive manufactured scaffolds. Here, scaffolds were fabricated using three polymers (polycaprolactone (PCL), poly(ethylene oxide)–poly(butylene terephthalate) (PEOT/PBT), and polylactic acid (PLA)) through additive manufacturing. Their chondrogenic and osteogenic potential were characterized and compared using human bone marrow‐derived mesenchymal stem cells (hBMSCs) through proteomics analysis. The 3D scaffolds were all hydrophilic and displayed Young's moduli close to those of bone or cartilage for PLA and PCL and PEOT/PBT, respectively. Biochemical assays indicated that PEOT/PBT and PLA scaffolds have a greater chondrogenic potential by higher glycosaminoglycan (GAG) and collagen deposition compared to PCL. PLA and PEOT/PBT showed to be more effective in promoting bone formation, as evidenced by higher calcium deposits detected by alizarin red staining, and higher alkaline phosphatase (ALP), especially for PLA in osteogenic medium. Proteomics data revealed the most distinct separation between conditions in chondrogenic medium, which had the highest protein identification rates. Pathway analysis showed that PCL did not induce any differentiation‐related pathways when compared to PEOT/PBT and PLA in any of the tested media conditions. Analysis of PEOT/PBT proteins showed pathways involved in chondrogenesis in all three media and pathways related to hypertrophic phenotype progression in chondrogenic medium. These data suggests that PEOT/PBT is a valuable candidate for cartilage and osteochondral applications, able to drive hBMSCs differentiation without the need of growth factors. PLA was also a valuable candidate for cartilage and bone applications by upregulating both chondrogenic and osteogenic‐related proteins in maintenance and chondrogenic media. In osteogenic and maintenance media, the upregulation of angiogenic proteins makes PLA a better candidate for bone application where vascularization is key.

Physioxia Expanded Bone Marrow Derived Mesenchymal Stem Cells Have Improved Cartilage Repair in an Early Osteoarthritic Focal Defect Model.

Focal early osteoarthritis (OA) or degenerative lesions account for 60% of treated cartilage defects each year. The current cell-based regenerative treatments have an increased failure rate for treating degenerative lesions compared to traumatic defects. Mesenchymal stem cells (MSCs) are an alternative cell source for treating early OA defects, due to their greater chondrogenic potential, compared to early OA chondrocytes. Low oxygen tension or physioxia has been shown to enhance MSC chondrogenic matrix content and could improve functional outcomes of regenerative therapies. The present investigation sought to develop a focal early OA animal model to evaluate cartilage regeneration and hypothesized that physioxic MSCs improve in vivo cartilage repair in both, post-trauma and focal early OA defects. Using a rabbit model, a focal defect was created, that developed signs of focal early OA after six weeks. MSCs cultured under physioxia had significantly enhanced in vitro MSC chondrogenic GAG content under hyperoxia with or without the presence of interleukin-1β (IL-1β). In both post-traumatic and focal early OA defect models, physioxic MSC treatment demonstrated a significant improvement in cartilage repair score, compared to hyperoxic MSCs and respective control defects. Future investigations will seek to understand whether these results are replicated in large animal models and the underlying mechanisms involved in in vivo cartilage regeneration.

Arthroscopic Debridement and Micronized Adipose Tissue Injection for End-Stage Ankle Arthritis

Category:Ankle, Ankle ArthritisIntroduction/Purpose:Joint arthritis is one of the leading musculoskeletal disorders in the adult population. The surgical treatment for end-stage ankle arthritis generally includes arthrodesis, distraction arthroplasty and total replacement. Recently, new therapeutic approaches, including the use of biologics, have been developed. Among the many sources, the adipose tissue is considered a greater chondrogenic potential and thus a lot of attention is being placed on it in the treatment of arthritis. Although several studies reported good clinical outcomes of adipose-based therapies for knee arthritis, there is a paucity of studies reporting the effects of adipose-based therapies for ankle arthritis. The purpose of this study is to evaluate the effect of intra- articular injection of autologous micronized adipose tissue in patients with end-stage ankle arthritis.Methods:A retrospective cohort study investigating consecutive patients treated with arthroscopic debridement and micronized adipose tissue injection for end-stage ankle arthritis was performed. Micronized adipose tissue was obtained from abdomen using a minimal manipulation technique in a closed system (Lipogems) without the addition of enzymes or any other additives. Clinical outcomes were evaluated using the Foot and Ankle Outcome Scores (FAOS), Short-Form 12 (SF-12), and visual analog scale (VAS) scores preoperatively and at final follow-up.Results:Total of 12 patients were included. The mean age was 57.5 years and mean follow-up time was 13.1 months. Mean symptoms, pain, daily activities and quality of life scores in FAOS significantly improved from 44.5, 41.3, 58.9, and 20.6 preoperatively to 76.2, 70.1, 80.9, and 60.7 postoperatively, respectively at final follow-up (p<0.001). The mean SF-12 and VAS score significantly improved (33.8 to 63.4, 7.2 to 2.9, respectively, p<0.001). One patient did not receive any benefits. In 4 patients, although they had symptomatic relief in early stage, FAOS and VAS scores deteriorated to baseline by 6 months after procedure. Total 4 patients were not satisfied with the procedures. No adverse events or relevant complications were reported.Conclusion:The current study demonstrates that arthroscopic debridement and micronized adipose tissue injection is a safe and beneficial procedure in the treatment of end-stage ankle arthritis. However, in 4 of 12 patients in this small cohort, the deterioration of clinical outcomes to the baseline was observed by 6 months after procedures. All of these patients had mechanical malalignment of hindfoot, suggesting that mechanical alignment may be one of the prognostic factors of this procedure. Further studies investigating longer-term effects in larger cohort should be warranted to confirm the benefit of micronized adipose tissue injection for end-stage ankle arthritis.

The comparative study of chondrogenic differentiation of mesenchymal stromal cells allocated from different sources

Introduction. As an alternative to autochondral transplantation, variants of chondrocyte replacement with mesenchymal stromal cells (MSCs) were considered, since these cells are present in all organs and tissues of human body and possess multilinear potential for differentiation. A number of studies demonstrate that the ability for chondrogenic differentiation of MSCs from different tissues varies, however, those studies are few and controversial. In accordance with the ethical principles and the technical ease of obtaining, adipose tissue, Wharton’s jelly (stroma) and dental pulp are the most attractive sources of MSCs for tissue engineering.Aim: to compare the chondrogenic potential of MSCs allocated from adipose tissue, Wharton’s jelly of the umbilical cord, and human deciduous teeth pulp cultured in the composition of microspheres (pellets).Materials and methods. The phenotype of primary cultures of MSCs was studied via flow microscopy. Chondrogenic differentiation was performed with 3D-culture in microspheres in the presence of TGFβ1 for two weeks under standard conditions. Human chondroblasts were used as a positive control. Cell viability was determined by fluorescent staining. Morphological study was performed using histological and immunohistochemical staining methods.Results. Cultures of MSCs from all sources had similar phenotypes CD29+, CD34–, CD44+, CD49b+, CD45–, CD73+, CD90+, HLADR. In the mass of living cells in microspheres only singular dead cells were visualized. A significant production of extracellular matrix (ECM) was observed in the chondroblast and adipose tissue microspheres, whereas in the microspheres of dental pulp and umbilical cord stroma derived MSCs, no significant ECM was detected. Among the MSCs, the highest production of collagen and glycosaminoglycans (GAG) in the ECM was observed in the microspheres of adipose tissue-derived MSCs, and the lowest production – in the microspheres of dental pulp-derived MSCs. Conclusion. A histological analysis of all microspheres after 14 days of culturing in the chondrogenic medium revealed the signs of differentiation in the chondrogenic direction, a progressive increase of the ECM produced by cells, and the presence of total collagen and GAG in it. Of all the MSCs studied, the greatest chondrogenic potential in vitro (the intensity of development of ECM components) is possessed by the adipose tissue-derived MSCs.

Adipose-Based Therapies for Knee Pain-Fat or Fiction.

Regenerative cell therapies are emerging as promising treatments for numerous musculoskeletal conditions, including knee osteoarthritis (OA). Adipose-derived stem cells and possibly other adipose-based therapies have a greater chondrogenic potential than stem cells derived from bone marrow, and thus a lot of attention is being placed on them as potential regenerative agents in the treatment of knee OA. Several types of adipose-based therapies have good basic science and preclinical data supporting their translation to human therapeutic intervention. Cultured, adipose-derived stem cells appear to be good source of bioactive cells with convenient accessibility, relative abundance, and well-documented regenerative capacity. Non-culture expanded adipose-based therapy, in the forms of stromal vascular fraction and most recently micronized adipose tissue (MAT), have been utilized in patients to treat OA and other cartilage abnormalities with encouraging preliminary data. These adipose-based therapies have shown a lot of therapeutic potential; however, because of the regulatory restrictions on enzymatic isolation and cell expansion, only MAT is currently available in clinical practice in the United States. While no serious adverse reactions have been reported, adipose-derived therapies also have the potential for adverse reactions including inflammation and infection. The current review provides an update on the latest research and presents this evidence on the therapeutic potential of adipose-based therapies in the treatment of knee OA.

Induced Redifferentiation of Human Chondrocytes from Articular Cartilage Lesion in Alginate Bead Culture After Monolayer Dedifferentiation: An Alternative Cell Source for Cell-Based Therapies?

Human chondrocytes isolated from articular cartilage (AC) lesions as an alternative cell source to the standard nonweight-bearing notch biopsy site may hold clinical potential for cell-based therapies. The aim was to characterize human AC lesion site chondrocytes, compare them to notch chondrocytes, and evaluate their redifferentiation potential after monolayer expansion and subsequent three-dimensional (3D) alginate bead culture. Lesion chondrocytes from knee joints of 20 patients with International Cartilage Repair Society (ICRS) grade 3 and 4 cartilage defects were analyzed ex vivo or cultured in primary alginate bead culture, monolayer expansion, or redifferentiated in alginate culture following monolayer expansion. The mRNA expression of the types I, II, and X collagen, and the proteoglycan aggrecan was compared between the four groups. In addition, notch chondrocytes of nine patients were compared to lesion chondrocytes ex vivo. AC lesion chondrocytes displayed ex vivo a nondegenerative phenotype, characterized by a relatively high mRNA expression of aggrecan and type II and X collagen, but a low type I collagen expression and a low ratio of type I to II collagen mRNA expression. Compared to notch chondrocytes, the mRNA expression of aggrecan and type II collagen was comparable and the ratio of type I to II collagen mRNA expression was below 1 in both groups, indicating a functional chondrocyte phenotype. Dedifferentiation led to a significantly altered degenerative mRNA expression profile. Induced redifferentiation in alginate beads after monolayer expansion significantly improved the mRNA expression of aggrecan, the type I and II collagen, and the type I to II collagen ratio, compared to monolayer expansion only. These data suggested that redifferentiating lesion chondrocytes after monolayer expansion in alginate beads resulted in a pool of cells with greater chondrogenic potential, compared to expanded dedifferentiated chondrocytes. Collectively, these data suggest that ex vivo and redifferentiated lesion chondrocytes may hold nonutilized clinical potential for the tissue engineering of AC.

Fetal Cartilage-Derived Cells Have Stem Cell Properties and Are a Highly Potent Cell Source for Cartilage Regeneration.

Current strategies for cartilage cell therapy are mostly based on the use of autologous chondrocytes or mesenchymal stem cells (MSCs). However, these cells have limitations of a small number of cells available and of low chondrogenic ability, respectively. Many studies now suggest that fetal stem cells are more plastic than adult stem cells and can therefore more efficiently differentiate into target tissues. However, the characteristics and the potential of progenitor cells from fetal tissue remain poorly defined. In this study, we examined cells from human fetal cartilage at 12 weeks after gestation in comparison with bone marrow-derived MSCs or cartilage chondrocytes from young donors (8-25 years old). The fetal cartilage-derived progenitor cells (FCPCs) showed higher yields by approximately 24 times than that of chondrocytes from young cartilage. The morphology of the FCPCs was polygonal at passage 0, being similar to that of the young chondrocytes, but it changed later at passage 5, assuming a fibroblastic shape more akin to that of MSCs. As the passages advanced, the FCPCs showed a much greater proliferation ability than the young chondrocytes and MSCs, with the doubling times ranging from 2∼4 days until passage 15. The surface marker profile of the FCPCs at passage 2 was quite similar to that of the MSCs, showing high expressions of CD29, CD90, CD105, and Stro-1. When compared to the young chondrocytes, the FCPCs showed much less staining of SA-β-gal, a senescence indicator, at passage 10 and no decrease in SOX9 expression until passage 5. They also showed a much greater chondrogenic potential than the young chondrocytes and the MSCs in a three-dimensional pellet culture in vitro and in polyglycolic acid (PGA) scaffolds in vivo. In addition, they could differentiate into adipogenic and osteogenic lineages as efficiently as MSCs in vitro. These results suggest that FCPCs have stem cell properties to some extent and that they are more active in terms of proliferation and chondrogenic differentiation than young chondrocytes or MSCs.

Human chondrocyte migration behaviour to guide the development of engineered cartilage.

Tissue-engineering techniques have been successful in developing cartilage-like tissues in vitro using cells from animal sources. The successful translation of these strategies to the clinic will likely require cell expansion to achieve sufficient cell numbers. Using a two-dimensional (2D) cell migration assay to first identify the passage at which chondrocytes exhibited their greatest chondrogenic potential, the objective of this study was to determine a more optimal culture medium for developing three-dimensional (3D) cartilage-like tissues using human cells. We evaluated combinations of commonly used growth factors that have been shown to promote chondrogenic growth and development. Human articular chondrocytes (AC) from osteoarthritic (OA) joints were cultured in 3D environments, either in pellets or encapsulated in agarose. The effect of growth factor supplementation was dependent on the environment, such that matrix deposition differed between the two culture systems. ACs in pellet culture were more responsive to bone morphogenetic protein (BMP2) alone or combinations containing BMP2 (i.e. BMP2 with PDGF or FGF). However, engineered cartilage development within agarose was better for constructs cultured with TGFβ3. These results with agarose and pellet culture studies set the stage for the development of conditions appropriate for culturing 3D functional engineered cartilage for eventual use in human therapies. Copyright © 2015 John Wiley & Sons, Ltd.

Combining freshly isolated chondroprogenitor cells from the infrapatellar fat pad with a growth factor delivery hydrogel as a putative single stage therapy for articular cartilage repair.

Growth factor delivery systems incorporating chondroprogenitor cells are an attractive potential treatment option for damaged cartilage. The rapid isolation, processing, and implantation of therapeutically relevant numbers of autologous chondroprogenitor cells, all performed "in-theatre" during a single surgical procedure, would significantly accelerate the clinical translation of such tissue engineered implants by avoiding the time, financial and regulatory challenges associated with in vitro cell expansion, and differentiation. The first objective of this study was to explore if rapid adherence to a specific substrate could be used as a simple means to quickly identify a subpopulation of chondroprogenitor cells from freshly digested infrapatellar fat pad (IFP) tissue. Adhesion of cells to tissue culture plastic within 30 min was examined as a mechanism of isolating subpopulations of cells from the freshly digested IFP. CD90, a cell surface marker associated with cell adhesion, was found to be more highly expressed in rapidly adhering cells (termed "RA" cells) compared to those that did not adhere (termed "NA" cells) in this timeframe. The NA subpopulation contained a lower number of colony forming cells, but overall had a greater chondrogenic potential but a diminished osteogenic potential compared to the RA subpopulation and unmanipulated freshly isolated (FI) control cells. When cultured in agarose hydrogels, NA cells proliferated faster than RA cells, accumulating significantly higher amounts of total sGAG and collagen. Finally, we sought to determine if cartilage tissue could be engineered by seeding such FI cells into a transforming growth factor-β3 delivery hydrogel. In such a system, both RA and NA cell populations demonstrated an ability to proliferate and produced a matrix rich in sGAG (∼2% w/w) that stained positively for type II collagen; however, the tissues were comparable to that generated using FI cells. Therefore, while the results of these in vitro studies do not provide strong evidence to support the use of selective substrate adhesion as a means to isolate chondroprogenitor cells, the findings demonstrate the potential of combining a growth factor delivery hydrogel and FI IFP cells as a single stage therapy for cartilage defect repair.

Therapeutic Superiority for Cartilage Repair by CD271-Positive Marrow Stromal Cell Transplantation

Recent reports indicated that human isolated CD271+ bone marrow mesenchymal stromal cells (BM-MSCs) have a greater expansion and potential for multipotent differentiation including chondrogenesis than classical plastic adherent (PA) BM-MSCs in vitro. Therefore, we set up a hypothesis that CD271+ MSCs may have a greater chondrogenic potential than PA-MSCs in vitro and in vivo. We investigated the superiority of CD271+ MSCs on chondrogenesis using in vitro expansion and pellet culture system and in vivo rat model of cartilage defect when compared to PA-MSCs. In the in vitro study, CD271+ MSCs showed higher expansion potential and produced larger pellets with higher expressions of chondrogenic genes when compared to the control groups. During the culture, CD271 expression decreased, which resulted in decreased chondrogenesis. In the in vivo study, immunohistochemical staining demonstrated differentiated human chondrocytes identified as double-stained cells with human-specific collagen type 2 and human leukocyte antigen-ABC in CD271+ and PA groups. The number of double-stained cells was significantly higher in the CD271+ group than PA group. Real-time RT-PCR analysis of tissue RNA isolated from the chondral defect site for human-specific chondrogenic markers demonstrated a significantly higher expression in CD271+ group than PA group. Macroscopic examination of chondral defect sites at week 8 revealed glossy white and well-integrated repaired tissues in the CD271+ and PA groups, but not in the PBS group. The average histological score in the CD271+ group was significantly greater than in the other groups. Apoptosis analysis at the cell transplanted site with TUNEL staining showed that the CD271+ group had significantly fewer apoptotic chondrocytes compared with the PA group. These results indicate that CD271+ MSCs have a greater chondrogenic potential than PA-MSCs in both in vitro and in vivo conditions.

Synovium-Derived Stem Cells: A Tissue-Specific Stem Cell for Cartilage Engineering and Regeneration

Articular cartilage is difficult to heal once injury or disease occurs. Autologous chondrocyte transplantation is a biological treatment with good prognosis, but donor site morbidity and limited cell source are disadvantages. Currently, mesenchymal stem cells (MSCs) are a promising approach for cartilage regeneration. Despite there being various sources, the best candidate for cartilage regeneration is the one with the greatest chondrogenic potential and the least hypertrophic differentiation. These properties are able to insure that the regenerated tissue is hyaline cartilage of high quality. This review article will summarize relevant literature to justify synovium-derived stem cells (SDSCs) as a tissue-specific stem cell for chondrogenesis by comparing synovium and cartilage with respect to anatomical location and functional structure, comparing the growth characterization and chondrogenic capacity of SDSCs and MSCs, evaluating the application of SDSCs in regenerative medicine and diseases, and discussing potential future directions.

Size of the embryoid body influences chondrogenesis of mouse embryonic stem cells

For applications in tissue engineering and regenerative medicine, embryonic stem cells (ESCs) are commonly pre-differentiated in the form of embryoid bodies (EBs). The uncontrolled cell differentiation in EBs results in a highly heterogeneous cell population, an unfavourable condition for therapeutic development. The purpose of this study was to determine an optimal size of EBs for chondrogenic differentiation. EBs were produced in suspension culture with mouse ESCs (ES-D3 GL). The 5-day-old EBs were sorted under a microscope by diameter: small EBs (S-EBs, < 100 microm), medium EBs (M-EBs, 100-150 microm) and large EBs (L-EBs, > 150 microm). The three sizes of EBs were cultured separately for 3 weeks in chondrogenic medium. Type II collagen and aggrecan gene expression was significantly upregulated in the S-EBs, when compared with the M-EBs and L-EBs (p < 0.05 and p < 0.001, respectively). Proteoglycans produced by the cells derived from S-EBs were > 50% of the other two groups. In addition, both Oct4 and Sox2 were expressed more in S-EBs than in M-EBs and L-EBs. Type X collagen expression was relatively increased in L-EBs. Slight shifts toward haematopoietic and endothelial differentiation were seen in the L- and M-EBs. In summary, the size of EBs has implications on ESC differentiation. Cells derived from S-EBs have a greater chondrogenic potential than those from M-EBs and L-EBs. The size of EBs can be a parameter utilized to optimize ESC differentiation for tissue engineering.

1095. Gene_Induced Chondrogenesis of Mesenchymal Stem Cells: A Comparative Analysis of Candidate cDNAs

Articular cartilage is a highly specialized tissue that protects the ends of bones in diarthrodal joints. The cartilage extracellular matrix is maintained by chondrocytes that occupy the tissue at low density. Because articular cartilage is avascular, it has a poor capacity to regenerate; thus, the damage resulting from large injuries is considered permanent. Due to their ready availability and capacity to differentiate along numerous lineages, adult mesenchymal stem cells (MSCs) may be useful in the development of cell-based therapies for cartilage repair. In this regard, gene transfer to MSCs can be used to achieve sustained expression of specific protein factors capable of inducing chondrogenic differentiation. In this study, using adenoviral mediated gene transfer and bone marrow derived MSCs in an aggregate culture system, we compared the relative condroinductive capacity of several cDNAs whose protein products have been associated with the induction or maintenance of the articular cartilage phenotype. First generation, E1, E3 deleted, serotype 5 adenoviral vectors carrying the complete cDNAs for TGF-b1, bone morphogenetic protein (BMP) -2, BMP -4 , BMP- 7, Sox 9, indian hedgehog (IHH), and insulin-like growth factor-1 (IGF-1) were constructed using cre-lox recombination. As a source of MSCs, adherent, colony forming cells from the bone marrow of 3-week-old calves were isolated, cultured and used at low passage. Individual flasks of MSCs were infected with Ad.TGF-b1, Ad.BMP-2, Ad.BMP-4, Ad.BMP-7 and Ad.IGF-1 at doses sufficient to provide low ( 100 ng/ml) amounts of transgene expression per 24 hrs. Delivery of Sox-9 and Ad.IHH was performed over a wide range of viral doses. At 24 hours post infection, 2.0 x 10^5 cells of each culture were pelleted by centrifugation. The aggregates were cultured for 3 weeks in conical tubes in a defined, serum-free medium containing ascorbate 2-phosphate and dexamethasone. Chondrogenesis was determined using histologic staining for glycosaminoglycans and by immunohistochemistry for collagen types I and II. By this method, gene transfer and expression of BMP-2 and BMP-4 showed the greatest chondrogenic potential. This was followed to a lesser extent by TGF-b1 and then Sox-9 and IGF-1. Delivery of IHH or BMP-7 was met with only minimal chondrogenic activity which was only slightly greater than uninfected control cells. The potent activity of the secreted factors BMPs-2, -4 and TGF-b1 were expected and are consistent with the literature. The capacity to induce chondrogenesis through delivery of the Sox-9 transcription factor offers a promising intracellular alternative to the overexpression of secretable pleiotropic growth factors. The relative lack of chondroinduction following overexpression of IGF-1 and IHH is perhaps not surprising because these products thought to be more important for maintenance than induction of the chondrocytic phenotype. Future work will involve evaluation these genes in combinatorial studies.

Differential behavior of anterior and posterior embryonic chick somites in vitro.

AbstractThe posterior somites of young (stages 15–18) chick embryos have a greater chondrogenic potential in tissue culture than do the anterior somites from the same embryo. Approximately 70% of posterior somite clusters will form cartilage nodules in tissue culture, whereas only 15–20% of anterior somite clusters form nodules. Paradoxically, when randomized anterior + posterior somites are cultured together, the incidence of cartilage formation is lower than expected. Randomized anterior + posterior somite clusters have the same incidence of cartilage formation as do anterior somites alone.Somites from either region, if cultured in the presence of embryonic notochord tissue, exhibit 100% cartilage formation.The chondrogenic potential of the somites may be correlated with their histo‐architecture and their embryonic history.