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1.
Eur Cell Mater ; 41: 153-169, 2021 02 10.
Article in English | MEDLINE | ID: mdl-33565057

ABSTRACT

Disc disease is characterised by degeneration of the nucleus pulposus (NP), the central gelatinous tissue of the intervertebral disc (IVD). As degeneration progresses, the microenvironment of the IVD becomes more hostile (i.e. decrease in oxygen, glucose and pH), providing a significant challenge for regeneration using cell-based therapies. Tissue engineering strategies such as priming cells or micro tissues with growth factors prior to implantation may overcome some of these issues by providing a pre-formed protective niche composed of extracellular matrix. The present study investigated the effect of priming on bone-marrow-derived stem cells (BMSCs) and articular chondrocytes (ACs) using transforming growth factor ß3 (TGF-ß3), cultured at different pH levels (pH 7.1, 6.8 and 6.5) representative of the in vivo disc microenvironment. Low pH was found to have a detrimental effect on both cell viability and matrix accumulation, which could be mitigated by priming cells using TGF-ß3. Investigating the activation of the transmembrane acid-sensing ion channels (ASIC-1 and -3) showed an increased expression of ASIC-1 in BMSCs and ASIC-3 in ACs at lower pH levels post-priming. Metabolic activity in terms of lactic acid production was also found to be affected significantly by priming, whereas oxygen and glucose consumptions did not change considerably. Overall, the study demonstrated that cells could be equipped to sustain the harsh environment of the IVD and promote accumulation of NP-like matrix through priming. Such an approach may open new avenues to engineer tissues capable of sustaining challenging microenvironments such as those found in the IVD.


Subject(s)
Intervertebral Disc Degeneration , Intervertebral Disc , Nucleus Pulposus , Extracellular Matrix , Humans , Hydrogen-Ion Concentration , Intervertebral Disc Degeneration/therapy
2.
Eur Cell Mater ; 37: 134-152, 2019 02 15.
Article in English | MEDLINE | ID: mdl-30768674

ABSTRACT

Priming towards a discogenic phenotype and subsequent cryopreservation of microencapsulated bone marrow stromal cells (BMSCs) may offer an attractive therapeutic approach for disc repair. It potentially obviates the need for in vivo administration of exogenous growth factors, otherwise required to promote matrix synthesis, in addition to providing 'off-the-shelf' availability. Cryopreserved and primed BMSC microcapsules were evaluated in an in vitro surrogate co-culture model system with nucleus pulposus (NP) cells under intervertebral disc (IVD)-like culture conditions and in an ex vivo bovine organ culture disc model. BMSCs were microencapsulated in alginate microcapsules and primed for 14 d with transforming growth factor beta-3 (TGF-ß3) under low oxygen conditions prior to cryopreservation. For the in vitro phase, BMSC microcapsules (unprimed or primed) were cultured for 28 d in a surrogate co-culture model system mimicking that of the IVD. For the ex vivo phase, microcapsules (unprimed or primed) were injected into the NP of bovine discs that underwent nucleotomy. In vitro results revealed that although NP cells produced significantly more matrix components in co-culture with BMSC microcapsules regardless of the differentiation state, unprimed microcapsules were inadequate at synthesising matrix as compared to primed microcapsules. However, this difference was diminished when evaluated in the ex vivo organ culture model,withboth unprimed and primed BMSC microcapsules accumulating large amounts of sulphated glycosaminoglycan (sGAG) and collagen and filling the defect cavity. Both models demonstrated that cryopreservation of BMSC microcapsules may offer a feasible strategy for predesigned delivery through cryobanking for on-demand regeneration of the IVD.


Subject(s)
Coculture Techniques/methods , Cryopreservation , Intervertebral Disc/physiology , Microspheres , Organ Culture Techniques/methods , Regeneration , Animals , Cattle , Cell Proliferation , Cell Separation , Cell Survival , Collagen/metabolism , DNA/metabolism , Glycosaminoglycans/metabolism , Stromal Cells/cytology , Swine
3.
Tissue Cell ; 49(4): 503-513, 2017 Aug.
Article in English | MEDLINE | ID: mdl-28515001

ABSTRACT

Chondrocyte based regenerative therapies for intervertebral disc repair such as Autologous Disc Cell Transplantation (ADCT, CODON) and allogeneic juvenile chondrocyte implantation (NuQu®, ISTO Technologies) have demonstrated good outcomes in clinical trials. However concerns remain with the supply demand reconciliation and issues surrounding immunoreactivity which exist for allogeneic-type technologies. The use of stem cells is challenging due to high growth factor requirements, regulatory barriers and differentiation towards a stable phenotype. Therefore, there is a need to identify alternative non-disc cell sources for the development and clinical translation of next generation therapies for IVD regeneration. In this study, we compared Nasal Chondrocytes (NC) as a non-disc alternative chondrocyte source with Articular Chondrocytes (AC) in terms of cell yield, morphology, proliferation kinetics and ability to produce key extracellular matrix components under 5% and 20% oxygen conditions, with and without exogenous TGF-ß supplementation. Results indicated that NC maintained proliferative capacity with high amounts of sGAG and lower collagen accumulation in the absence of TGF-ß supplementation under 5% oxygen conditions. Importantly, osteogenesis and calcification was inhibited for NC when cultured in IVD-like microenvironmental conditions. The present study provides a rationale for the exploration of nasal chondrocytes as a promising, potent and clinically feasible autologous cell source for putative IVD repair strategies.


Subject(s)
Cartilage, Articular , Chondrocytes , Extracellular Matrix/metabolism , Intervertebral Disc , Nasal Cartilages , Regenerative Medicine/methods , Animals , Cartilage, Articular/cytology , Cartilage, Articular/metabolism , Cattle , Cells, Cultured , Chondrocytes/cytology , Chondrocytes/metabolism , Chondrocytes/transplantation , Female , Nasal Cartilages/cytology , Nasal Cartilages/metabolism
4.
Acta Biomater ; 36: 55-62, 2016 05.
Article in English | MEDLINE | ID: mdl-26961807

ABSTRACT

UNLABELLED: Freshly isolated stromal cells can potentially be used as an alternative to in vitro expanded cells in regenerative medicine. Their use requires the development of bioactive hydrogels or scaffolds which provide an environment to enhance their proliferation and tissue-specific differentiation in vivo. The goal of the current study was to develop an injectable fibrin hydrogel functionalized with cartilage ECM microparticles and transforming growth factor (TGF)-ß3 as a putative therapeutic for articular cartilage regeneration. ECM microparticles were produced by cryomilling and freeze-drying porcine articular cartilage. Up to 2% (w/v) ECM could be incorporated into fibrin without detrimentally affecting its capacity to form stable hydrogels. To access the chondroinductivity of cartilage ECM, we compared chondrogenesis of infrapatellar fat pad-derived stem cells in fibrin hydrogels functionalized with either particulated ECM or control gelatin microspheres. Cartilage ECM particles could be used to control the delivery of TGF-ß3 to IFP-derived stem cells within fibrin hydrogels in vitro, and furthermore, led to higher levels of sulphated glycosaminoglycan (sGAG) and collagen accumulation compared to control constructs loaded with gelatin microspheres. In vivo, freshly isolated stromal cells generated a more cartilage-like tissue within fibrin hydrogels functionalized with cartilage ECM particles compared to the control gelatin loaded constructs. These tissues stained strongly for type II collagen and contained higher levels of sGAGs. These results support the use of fibrin hydrogels functionalized with cartilage ECM components in single-stage, cell-based therapies for joint regeneration. STATEMENT OF SIGNIFICANCE: An alternative to the use of in vitro expanded cells in regenerative medicine is the use of freshly isolated stromal cells, where a bioactive scaffold or hydrogel is used to provide an environment that enhances their proliferation and tissue-specific differentiation in vivo. The objective of this study was to develop an injectable fibrin hydrogel functionalized with cartilage ECM micro-particles and the growth factor TGF-ß3 as a therapeutic for articular cartilage regeneration. This study demonstrates that freshly isolated stromal cells generate cartilage tissue in vivo when incorporated into such a fibrin hydrogels functionalized with cartilage ECM particles. These findings open up new possibilities for in-theatre, single-stage, cell-based therapies for joint regeneration.


Subject(s)
Cartilage/physiology , Chondrogenesis , Extracellular Matrix/chemistry , Fibrin/chemistry , Hydrogels/chemistry , Regeneration , Animals , Cartilage/cytology , Female , Humans , Male , Stromal Cells/cytology , Stromal Cells/metabolism , Swine
5.
Eur Cell Mater ; 30: 163-85; discussion 185-6, 2015 Sep 28.
Article in English | MEDLINE | ID: mdl-26412388

ABSTRACT

Arthroplasty is currently the only surgical procedure available to restore joint function following articular cartilage and bone degeneration associated with diseases such as osteoarthritis (OA). A potential alternative to this procedure would be to tissue-engineer a biological implant and use it to replace the entire diseased joint. The objective of this study was therefore to tissue-engineer a scaled-up, anatomically shaped, osteochondral construct suitable for partial or total resurfacing of a diseased joint. To this end it was first demonstrated that a bone marrow derived mesenchymal stem cell seeded alginate hydrogel could support endochondral bone formation in vivo within the osseous component of an osteochondral construct, and furthermore, that a phenotypically stable layer of articular cartilage could be engineered over this bony tissue using a co-culture of chondrocytes and mesenchymal stem cells. Co-culture was found to enhance the in vitro development of the chondral phase of the engineered graft and to dramatically reduce its mineralisation in vivo. In the final part of the study, tissue-engineered grafts (~ 2 cm diameter) mimicking the geometry of medial femorotibial joint prostheses were generated using laser scanning and rapid prototyped moulds. After 8 weeks in vivo, a layer of cartilage remained on the surface of these scaled-up engineered implants, with evidence of mineralisation and bone development in the underlying osseous region of the graft. These findings open up the possibility of a tissue-engineered treatment option for diseases such as OA.


Subject(s)
Bone and Bones/cytology , Chondrocytes/cytology , Knee Joint/cytology , Osteogenesis/physiology , Tissue Engineering , Tissue Scaffolds , Cartilage, Articular/cytology , Chondrogenesis/physiology , Coculture Techniques/methods , Knee Joint/pathology , Mesenchymal Stem Cells/cytology , Tissue Engineering/methods
6.
J Biomech ; 47(9): 2115-21, 2014 Jun 27.
Article in English | MEDLINE | ID: mdl-24377681

ABSTRACT

The objective of this study was to investigate how joint specific biomechanical loading influences the functional development and phenotypic stability of cartilage grafts engineered in vitro using stem/progenitor cells isolated from different source tissues. Porcine bone marrow derived multipotent stromal cells (BMSCs) and infrapatellar fat pad derived multipotent stromal cells (FPSCs) were seeded in agarose hydrogels and cultured in chondrogenic medium, while simultaneously subjected to 10MPa of cyclic hydrostatic pressure (HP). To mimic the endochondral phenotype observed in vivo with cartilaginous tissues engineered using BMSCs, the culture media was additionally supplemented with hypertrophic factors, while the loss of phenotype observed in vivo with FPSCs was induced by withdrawing transforming growth factor (TGF)-ß3 from the media. The application of HP was found to enhance the functional development of cartilaginous tissues engineered using both BMSCs and FPSCs. In addition, HP was found to suppress calcification of tissues engineered using BMSCs cultured in chondrogenic conditions and acted to maintain a chondrogenic phenotype in cartilaginous grafts engineered using FPSCs. The results of this study point to the importance of in vivo specific mechanical cues for determining the terminal phenotype of chondrogenically primed multipotent stromal cells. Furthermore, demonstrating that stem or progenitor cells will appropriately differentiate in response to such biophysical cues might also be considered as an additional functional assay for evaluating their therapeutic potential.


Subject(s)
Adipose Tissue/cytology , Bone Marrow Cells/cytology , Cartilage , Stromal Cells/cytology , Tissue Engineering , Animals , Cell Differentiation , Cells, Cultured , Chondrogenesis , Hydrostatic Pressure , Phenotype , Swine
7.
Equine Vet J ; 46(4): 494-8, 2014 Jul.
Article in English | MEDLINE | ID: mdl-24004343

ABSTRACT

REASONS FOR PERFORMING STUDY: Selection of suture material in equine surgery is often based on costs or subjective factors, such as the surgeon's personal experience, rather than objective facts. The amount of objective data available on durability of suture materials with regard to specific equine physiological conditions is limited. OBJECTIVES: To evaluate the effect of various equine physiological and pathological fluids on the rate of degradation of a number of commonly used suture materials. STUDY DESIGN: In vitro material testing. METHODS: Suture materials were exposed in vitro to physiological fluid, followed by biomechanical analysis. Three absorbable suture materials, glycolide/lactide copolymer, polyglactin 910 and polydioxanone were incubated at 37°C for 7, 14 or 28 days in phosphate-buffered saline, equine serum, equine urine and equine peritoneal fluid from an animal with peritonitis. Five strands of each suture material type were tested to failure in a materials testing machine for each time point and each incubation medium. Yield strength, strain and Young's modulus were calculated, analysed and reported. RESULTS: For all suture types, the incubation time had a significant effect on yield strength, percentage elongation and Young's modulus in all culture media (P<0.0001). Suture type was also shown significantly to influence changes in each of yield strength, percentage elongation and Young's modulus in all culture media (P<0.0001). While the glycolide/lactide copolymer demonstrated the highest Day 0 yield strength, it showed the most rapid degradation in all culture media. For each of the 3 material characteristics tested, polydioxanone showed the least variation across the incubation period in each culture medium. CONCLUSIONS: The duration of incubation and the type of fluid have significant effects on the biomechanical properties of various suture materials. These findings are important for evidence-based selection of suture material in clinical cases.


Subject(s)
Body Fluids/chemistry , Horses , Materials Testing/veterinary , Sutures/veterinary , Animals , Elasticity , Equipment Failure Analysis
8.
J Mech Behav Biomed Mater ; 11: 102-11, 2012 Jul.
Article in English | MEDLINE | ID: mdl-22658159

ABSTRACT

MSCs from non-cartilaginous knee joint tissues such as the infrapatellar fat pad (IFP) and synovium possess significant chondrogenic potential and provide a readily available and clinically feasible source of chondroprogenitor cells. Fibroblast growth factor-2 (FGF-2) has been shown to be a potent mitotic stimulator during ex vivo expansion of MSCs, as well as regulating their subsequent differentiation potential. The objective of this study was to investigate the longer term effects of FGF-2 expansion on the functional development of cartilaginous tissues engineered using MSCs derived from the IFP. IFP MSCs were isolated and expanded to passage 2 in a standard media formulation with or without FGF-2 (5 ng/ml) supplementation. Expanded cells were encapsulated in agarose hydrogels, maintained in chondrogenic media for 42 days and analysed to determine their mechanical properties and biochemical composition. Culture media, collected at each feed, was also analysed for biochemical constituents. MSCs expanded in the presence of FGF-2 proliferated more rapidly, with higher cell yields and lower population doubling times. FGF-2 expanded MSCs generated the most mechanically functional tissue. Matrix accumulation was dramatically higher after 21 days for FGF-2 expanded MSCs, but decreased between day 21 and 42. By day 42, FGF-2 expanded MSCs had still accumulated ∼1.4 fold higher sGAG and ∼1.7 fold higher collagen compared to control groups. The total amount of sGAG synthesised (retained in hydrogels and released into the media) was ∼2.4 fold higher for FGF-2 expanded MSCs, with only ∼25% of the total amount generated being retained within the constructs. Further studies are required to investigate whether IFP derived MSCs have a diminished capacity to synthesise other matrix components important in the aggregation, assembly and retention of proteoglycans. In conclusion, expanding MSCs in the presence of FGF-2 rapidly accelerates chondrogenesis in 3D agarose cultures resulting in superior mechanical functionality.


Subject(s)
Adipose Tissue/cytology , Cartilage/cytology , Fibroblast Growth Factor 2/pharmacology , Mesenchymal Stem Cells/cytology , Mesenchymal Stem Cells/drug effects , Patella/cytology , Tissue Engineering , Animals , Cell Proliferation/drug effects , Glycosaminoglycans/metabolism , Hydrogels/pharmacology , Mesenchymal Stem Cells/metabolism , Sepharose/pharmacology , Time Factors
9.
Acta Biomater ; 8(6): 2153-9, 2012 Jul.
Article in English | MEDLINE | ID: mdl-22426136

ABSTRACT

Both hydrostatic pressure (HP) and cell-matrix interactions have independently been shown to regulate the chondrogenic differentiation of mesenchymal stem cells (MSCs). The objective of this study was to test the hypothesis that the response of MSCs to hydrostatic pressure will depend on the biomaterial within which the cells are encapsulated. Bone-marrow-derived MSCs were seeded into either agarose or fibrin hydrogels and exposed to 10 MPa of cyclic HP (1 Hz, 4 h per day, 5 days per week for 3 weeks) in the presence of either 1 or 10 ng ml(-1) of TGF-ß3. Agarose hydrogels were found to support a spherical cellular morphology, while MSCs seeded into fibrin hydrogels attached and spread, with clear stress fiber formation. Hydrogel contraction was also observed in MSC-fibrin constructs. While agarose hydrogels better supported chondrogenesis of MSCs, HP only enhanced sulfated glycosaminoglycan (sGAG) accumulation in fibrin hydrogels, which correlated with a reduction in fibrin contraction. HP also reduced alkaline phosphatase activity in the media for both agarose and fibrin constructs, suggesting that this stimulus plays a role in the maintenance of the chondrogenic phenotype. This study demonstrates that a complex relationship exists between cell-matrix interactions and hydrostatic pressure, which plays a key role in regulating the chondrogenic differentiation of MSCs.


Subject(s)
Extracellular Matrix , Mesenchymal Stem Cells/cytology , Animals , Cell Proliferation , Cells, Cultured , Fibrin , Immunohistochemistry , Microscopy, Confocal , Sepharose , Swine
10.
J Tissue Eng Regen Med ; 5(9): 673-83, 2011 Oct.
Article in English | MEDLINE | ID: mdl-21953865

ABSTRACT

The objective of this study was to determine the functional properties of cartilaginous tissues generated by porcine MSCs isolated from different tissue sources, and to compare these properties to those derived from chondrocytes (CCs). MSCs were isolated from bone marrow (BM) and infrapatellar fat pad (FP), while CCs were harvested from the articular surface of the femoro-patellar joint. Culture-expanded CCs and MSCs were encapsulated in agarose hydrogels and cultured in the presence of TGFß3. Samples were analysed biomechanically, biochemically and histologically at days 0, 21 and 42. After 42 days in free swelling culture, mean GAG content was 1.50% w/w in CC-seeded constructs, compared to 0.95% w/w in FP- and 0.43% w/w in BM-seeded constructs. Total collagen accumulation was highest in FP constructs. DNA content increased with time for all the groups. The mechanical functionality of cartilaginous tissues engineered using CCs was superior to that generated from either source of MSCs. Differences were also observed in the spatial distribution of matrix components in tissues engineered using CCs and MSCs, which appears to have a strong influence on the apparent mechanical properties of the constructs. Therefore, while functional cartilaginous tissues can be engineered using MSCs isolated from different sources, the spatial composition of these tissues is unlike that generated using chondrocytes, suggesting that MSCs and chondrocytes respond differently to the regulatory factors present within developing cartilaginous constructs.


Subject(s)
Adipose Tissue/cytology , Bone Marrow Cells/cytology , Cartilage/physiology , Chondrocytes/cytology , Mesenchymal Stem Cells/cytology , Patella/cytology , Tissue Engineering/methods , Animals , Cell Separation , Cells, Cultured , Collagen/metabolism , DNA/metabolism , Elastic Modulus , Glycosaminoglycans/metabolism , Hydrogel, Polyethylene Glycol Dimethacrylate , Multipotent Stem Cells/cytology , Osteogenesis , Sepharose , Structure-Activity Relationship , Sus scrofa
11.
J Mech Behav Biomed Mater ; 4(7): 1257-65, 2011 Oct.
Article in English | MEDLINE | ID: mdl-21783134

ABSTRACT

Mechanical signals can play a key role in regulating the chondrogenic differentiation of mesenchymal stem cells (MSCs). The objective of this study was to determine if the long-term application of cyclic hydrostatic pressure could be used to improve the functional properties of cartilaginous tissues engineered using bone marrow derived MSCs. MSCs were isolated from the femora of two porcine donors, expanded separately under identical conditions, and then suspended in cylindrical agarose hydrogels. Constructs from both donors were maintained in a chemically defined media supplemented with TGF-ß3 for 42 days. TGF-ß3 was removed from a subset of constructs from day 21 to 42. Loaded groups were subjected to 10 MPa of cyclic hydrostatic pressurisation at 1 Hz for one hour/day, five days/week. Loading consisted either of continuous hydrostatic pressure (CHP) initiated at day 0, or delayed hydrostatic pressure (DHP) initiated at day 21. Free swelling (FS) constructs were cultured in parallel as controls. Constructs were assessed at days 0, 21 and 42. MSCs isolated from both donors were morphologically similar, demonstrated comparable colony forming unit-fibroblast (CFU-F) numbers, and accumulated near identical levels of collagen and GAG following 42 days of free swelling culture. Somewhat unexpectedly the two donors displayed a differential response to hydrostatic pressure. For one donor the application of CHP resulted in increased collagen and GAG accumulation by day 42, resulting in an increased dynamic modulus compared to FS controls. In contrast, CHP had no effect on matrix accumulation for the other donor. The application of DHP had no effect on either matrix accumulation or construct mechanical properties for both donors. Variability in the response to hydrostatic pressure was also observed for three further donors. In conclusion, this study demonstrates that the application of long-term hydrostatic pressure can be used to improve the functional properties of cartilaginous tissues engineered using bone marrow derived MSCs by enhancing collagen and GAG accumulation. The response to such loading however is donor dependent, which has implications for the clinical utilisation of such a stimulus when engineering cartilaginous grafts using autologous MSCs.


Subject(s)
Bone Marrow Cells/cytology , Cartilage/cytology , Mesenchymal Stem Cells/cytology , Tissue Engineering/methods , Animals , Biomechanical Phenomena , Cartilage/drug effects , Cartilage/growth & development , Cartilage/metabolism , Cell Differentiation/drug effects , Chondrogenesis/drug effects , Collagen/metabolism , Elastic Modulus , Glycosaminoglycans/metabolism , Humans , Hydrogels/chemistry , Hydrostatic Pressure , Mesenchymal Stem Cells/drug effects , Mesenchymal Stem Cells/metabolism , Sepharose/chemistry , Swine , Transforming Growth Factor beta3/pharmacology
12.
J Bone Joint Surg Br ; 93(4): 517-24, 2011 Apr.
Article in English | MEDLINE | ID: mdl-21464493

ABSTRACT

The scarcity of mesenchymal stem cells (MSCs) in iliac crest bone marrow aspirate (ICBMA), and the expense and time in culturing cells, has led to the search for alternative harvest sites. The reamer-irrigation-aspirator (RIA) provides continuous irrigation and suction during reaming of long bones. The aspirated contents pass via a filter, trapping bony fragments, before moving into a 'waste' bag from which MSCs have been previously isolated. We examined the liquid and solid phases, performed a novel digestion of the solid phase, and made a comparative assessment in terms of number, phenotype and differentiation capacity with matched ICBMA. The solid fraction from the filtrate was digested for 60 minutes at 37° C with collagenase. Enumeration was performed via the colony-forming unit fibroblast (CFU-F) assay. Passage (P2) cells were differentiated towards osteogenic, adipogenic and chondrogenic lineages, and their phenotypes assessed using flow cytometry (CD33, CD34, CD45, CD73, CD90, and CD105). MSCs from the RIA phases were able to differentiate at least as well as those from ICBMA, and all fractions had phenotypes consistent with other established sources. The median number of colonies for the three groups was: ICBMA = 8.5 (2 to 86), RIA-liquid = 19.5 (4 to 90), RIA-solid = 109 (67 to 200) per 200 µl. The mean total yield of cells for the three groups was: ICBMA = 920 (0 to 4275), RIA-liquid = 114,983 (16,500 to 477,750), RIA-solid = 12,785 (7210 to 28 475). The RIA filtrate contains large numbers of MSCs that could potentially be extracted without enzymatic digestion and used for bone repair without prior cell expansion.


Subject(s)
Bone Marrow Cells/cytology , Cell Separation/methods , Mesenchymal Stem Cells/cytology , Adult , Bone Regeneration/physiology , Cell Culture Techniques , Cell Differentiation/physiology , Cells, Cultured , Equipment Design , Female , Humans , Male , Middle Aged
13.
Osteoarthritis Cartilage ; 18(10): 1345-54, 2010 Oct.
Article in English | MEDLINE | ID: mdl-20650328

ABSTRACT

BACKGROUND: For current tissue engineering or regenerative medicine strategies, chondrocyte (CC)- or mesenchymal stem cell (MSC)-seeded constructs are typically cultured in normoxic conditions (20% oxygen). However, within the knee joint capsule a lower oxygen tension exists. OBJECTIVE: The objective of this study was to investigate how CCs and infrapatellar fad pad derived MSCs will respond to a low oxygen (5%) environment in 3D agarose culture. Our hypothesis was that culture in a low oxygen environment (5%) will enhance the functional properties of cartilaginous tissues engineered using both cell sources. EXPERIMENTAL DESIGN: Cell-encapsulated agarose hydrogel constructs (seeded with CCs or infrapatellar fat pad (IFP) derived MSCs) were prepared and cultured in a chemically defined serum-free medium in the presence (CCs and MSCs) or absence (CCs only) of transforming growth factor-beta3 (TGF-ß3) in normoxic (20%) or low oxygen (5%) conditions for 42 days. Constructs were assessed at days 0, 21 and 42 in terms of mechanical properties, biochemical content and histologically. RESULTS: Low oxygen tension (5%) was observed to promote extracellular matrix (ECM) production by CCs cultured in the absence of TGF-ß3, but was inhibitory in the presence of TGF-ß3. In contrast, a low oxygen tension enhanced chondrogenesis of IFP constructs in the presence of TGF-ß3, leading to superior mechanical functionality compared to CCs cultured in identical conditions. CONCLUSIONS: Extrapolating the results of this study to the in vivo setting, it would appear that joint fat pad derived MSCs may possess a superior potential to generate a functional repair tissue in low oxygen tensions. However, in the context of in vitro cartilage tissue engineering, CCs maintained in normoxic conditions in the presence of TGF-ß3 generate the most mechanically functional tissue.


Subject(s)
Cartilage, Articular/cytology , Cell Hypoxia/physiology , Chondrocytes/cytology , Mesenchymal Stem Cells/cytology , Tissue Engineering/methods , Adipose Tissue/cytology , Animals , Cartilage, Articular/physiology , Cells, Cultured , Chondrocytes/drug effects , Chondrocytes/physiology , Chondrogenesis/physiology , Collagen/metabolism , Extracellular Matrix/metabolism , Hydrogel, Polyethylene Glycol Dimethacrylate , Mesenchymal Stem Cells/physiology , Sus scrofa , Transforming Growth Factor beta3/pharmacology
14.
Ann Biomed Eng ; 37(12): 2556-65, 2009 Dec.
Article in English | MEDLINE | ID: mdl-19757060

ABSTRACT

Integration of repair tissue is a key indicator of the long-term success of cell-based therapies for cartilage repair. The objective of this study was to compare the in vitro chondrogenic differentiation and integration of agarose hydrogels seeded with either chondrocytes or bone marrow-derived mesenchymal stem cells (MSCs) in defects created in cartilage explants. Chondrocytes and MSCs were isolated from porcine donors, suspended in 2% agarose and then injected into cylindrical defects within the explants. These constructs were maintained in a chemically defined medium supplemented with 10 ng/mL of TGF-beta3. Cartilage integration was assessed by histology and mechanical push-out tests. After 6 weeks in culture, chondrocyte-seeded constructs demonstrated a higher integration strength (64.4 +/- 8.3 kPa) compared to MSC-seeded constructs (22.7 +/- 5.9 kPa). Glycosaminoglycan (GAG) (1.27 +/- 0.3 vs. 0.19 +/- 0.03 kPa) and collagen (0.31 +/- 0.08 vs. 0.09 +/- 0.01 kPa) accumulation in chondrocyte-seeded constructs was greater than that measured in the MSC-seeded group. The GAG, collagen, and DNA content of both chondrocyte- and MSC-seeded hydrogels cultured in cartilage explants was significantly lower than control constructs cultured in free swelling conditions. The results of this study suggest that the explant model may constitute a more rigorous in vitro test to assess MSC therapies for cartilage defect repair.


Subject(s)
Cartilage, Articular/injuries , Cartilage, Articular/surgery , Chondrocytes/physiology , Chondrocytes/transplantation , Chondrogenesis , Mesenchymal Stem Cell Transplantation/methods , Animals , Cartilage, Articular/pathology , Cells, Cultured , Chondrocytes/cytology , In Vitro Techniques , Swine
15.
Biochem Biophys Res Commun ; 377(2): 458-462, 2008 Dec 12.
Article in English | MEDLINE | ID: mdl-18851955

ABSTRACT

The objective of this study was to investigate the influence of dynamic compressive loading on chondrogenesis of mesenchymal stem cells (MSCs) in the presence of TGF-beta3. Isolated porcine MSCs were suspended in 2% agarose and subjected to intermittent dynamic compression (10% strain) for a period of 42 days in a dynamic compression bioreactor. After 42 days in culture, the free-swelling specimens exhibited more intense alcian blue staining for proteoglycans, while immunohistochemical analysis revealed increased collagen type II immunoreactivity. Glycosaminoglycan (GAG) content increased with time for both free-swelling and dynamically loaded constructs, and by day 42 it was significantly higher in both the core (2.5+/-0.21%w/w vs. 0.94+/-0.03%w/w) and annulus (1.09+/-0.09%w/w vs. 0.59+/-0.08%w/w) of free-swelling constructs compared to dynamically loaded constructs. This result suggests that further optimization is required in controlling the biomechanical and/or the biochemical environment if such stimuli are to have beneficial effects in generating functional cartilaginous tissue.


Subject(s)
Chondrogenesis , Compressive Strength , Mesenchymal Stem Cells/cytology , Animals , Bioreactors , Cell Culture Techniques , Chondrogenesis/drug effects , Chondrogenesis/physiology , Collagen Type II/metabolism , Glycosaminoglycans/metabolism , Mesenchymal Stem Cells/drug effects , Mesenchymal Stem Cells/physiology , Swine , Transforming Growth Factor beta3/pharmacology
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