A Novel Method Facilitating the Simple and Low-Cost Preparation of Human Osteochondral Slice Explants for Large-Scale Native Tissue Analysis.

For in vitro modeling of human joints, osteochondral explants represent an acceptable compromise between conventional cell culture and animal models. However, the scarcity of native human joint tissue poses a challenge for experiments requiring high numbers of samples and makes the method rather unsuitable for toxicity analyses and dosing studies. To scale their application, we developed a novel method that allows the preparation of up to 100 explant cultures from a single human sample with a simple setup. Explants were cultured for 21 days, stimulated with TNF-α or TGF-ß3, and analyzed for cell viability, gene expression and histological changes. Tissue cell viability remained stable at >90% for three weeks. Proteoglycan levels and gene expression of COL2A1, ACAN and COMP were maintained for 14 days before decreasing. TNF-α and TGF-ß3 caused dose-dependent changes in cartilage marker gene expression as early as 7 days. Histologically, cultures under TNF-α stimulation showed a 32% reduction in proteoglycans, detachment of collagen fibers and cell swelling after 7 days. In conclusion, thin osteochondral slice cultures behaved analogously to conventional punch explants despite cell stress exerted during fabrication. In pharmacological testing, both the shorter diffusion distance and the lack of need for serum in the culture suggest a positive effect on sensitivity. The ease of fabrication and the scalability of the sample number make this manufacturing method a promising platform for large-scale preclinical testing in joint research.

An assessment of bone marrow mesenchymal stem cell and human articular cartilage derived chondroprogenitor cocultures vs. monocultures.

BACKGROUND: Cell based therapy in cartilage repair predominantly involves the use of chondrocytes and mesenchymal stromal cells (MSC). Co-culture systems, due to their probable synergistic effect on enhancement of functional chondrogenesis and reduction in terminal differentiation have also been attempted. Chondroprogenitors, derived from articular cartilage and regarded as MSCs, have recently garnered interest for consideration in cartilage regeneration to overcome limitations associated with use of conventional cell types. The aim of this study was to assess whetherco-culturing bone marrow (BM)-MSCs and chondroprogenitors at different ratios would yield superior results in terms of surface marker expression, gene expression and chondrogenic potential. METHODS: Human BM-MSCs and chondroprogenitors obtained from three osteoarthritic knee joints and subjected to monolayer expansion and pellet cultures (10,000 cells/cm2) as five test groups containing either monocultures or co-cultures (MSC: chondroprogenitors) at three different ratios (75:25, 50:50 and 25:75) were utilized. RESULTS: Data analysis revealed that all groups exhibited a high expression of CD166, CD29 and CD49e. With regard to gene expression, high expression of SOX9, Aggrecan and Collagen type I; a moderate expression of Collagen type X and RUNX2; with a low expression of Collagen type II was seen. Analysis of pellet culture revealed that chondroprogenitor monoculture and chondroprogenitor dominant coculture, exhibited a subjectively larger pellet size with higher deposition of Collagen type II and glycosaminoglycan. CONCLUSION: In conclusion, this study is suggestive of chondroprogenitor monoculture superiority over MSCs, either in isolation or in a coculture system and proposes further analysis of chondroprogenitors for cartilage repair.

Assessment of cartilage regeneration on 3D collagen-polycaprolactone scaffolds: Evaluation of growth media in static and in perfusion bioreactor dynamic culture.

Efforts on bioengineering are directed towards the construction of biocompatible scaffolds and the determination of the most favorable microenvironment, which will better support cell proliferation and differentiation. Perfusion bioreactors are attracting growing attention as an effective, modern tool in tissue engineering. A natural biomaterial extensively used in regenerative medicine with outstanding biocompatibility, biodegradability and non-toxic characteristics, is collagen, a structural protein with undisputed beneficial characteristics. This is a study designed according to the above considerations. 3D printed polycaprolactone (PCL) scaffolds with rectangular pores were coated with collagen either as a coating on the scaffold's trabeculae, or as a gel-cell solution penetrating scaffolds' pores. We employed histological, molecular and imaging techniques to analyze colonization, proliferation and chondrogenic differentiation of Adipose Derived Mesenchymal Stem Cells (ADMSCs). Two different differentiation culture media were employed to test chondrogenic differentiation on gelated and non gelated PCL scaffolds in static and in perfusion bioreactors dynamic culture conditions. In dynamic culture, non gelated scaffolds combined with our in house TGF-ß2 based medium, augmented chondrogenic differentiation performance, which overall was significantly less favorable compared to StemPro™ propriety medium. The beneficial mechanical stimulus of dynamic culture, appears to outgrow the disadvantage of the "weaker" TGF-ß2 medium used for chondrogenic differentiation. Even though cells in static culture grew well on the scaffold, there was limited penetration inside the construct, so the purpose of the 3D culture was not fully served. In contrast dynamic culture achieved better penetration and uniform distribution of the cells within the scaffold.

Diffusion-weighted 7.0T Magnetic Resonance Imaging in Assessment of Intervertebral Disc Degeneration in Rats.

BACKGROUND: Intervertebral disc degeneration (IDD) is a major cause of disc protrusion, likely to be associated with decrease of water content. This research aimed to evaluate IDD by diffusion-weighted imaging (DWI) with a 7.0 Tesla (T) magnetic resonance imaging (MRI) machine. METHODS: A total of 24 healthy Sprague-Dawley rats were randomly selected and divided into four groups (A, B, C, and D), each consisting of 3 male and 3 female rats (28, 42, 56, and 70 days old, respectively). All the rats were imaged with a 7.0T MRI, producing T2WI, T1WI, and functional DWI sequences. Data were collected and apparent diffusion coefficient (ADC) charts were constructed. Nucleus pulposus and annulus fibrosus regions were identified, several regions of interest were chosen, and their ADC values were obtained. After imaging, rats were sacrificed and their intervertebral discs (L1-L6) were dissected, yielding a total of 144 discs. Protein was extracted for the purpose of Western blotting. Comparison among multiple samples used one-way analysis of variance and least significant difference methods. RESULTS: 7.0T MRI revealed evident decrease in signal intensity within intervertebral discs of Sprague-Dawley rats with age. Intervertebral disc ADC values significantly decreased from Group A (0.00154 ± 0.00008) to Group D (0.00107 ± 0.00007; P < 0.01); nucleus pulposus ADC values significantly decreased from Group A (0.00164 ± 0.00005) to Group D (0.00140 ± 0.00007; P < 0.01) and annulus fibrosus ADC values significantly decreased from Group A (0.00129 ± 0.00014) to Group D (0.00082 ± 0.00012; P < 0.01). Meanwhile, it also revealed evident decrease from high spinal level to low spinal level: nucleus pulposus ADC values in Group A significantly decreased from L1/L2 (0.00163 ± 0.00006) to L6/S1 (0.00139 ± 0.00004; P < 0.01). While annulus fibrosus ADC values did not differ significantly between levels in Group A (P > 0.05). Western blotting showed that aggrecan content of intervertebral discs decreased from Group A (1.88 ± 0.16) to Group D (0.17 ± 0.04) with age (P < 0.01); Type II collagen content of intervertebral discs decreased from Group A (2.22 ± 0.04) to Group D (0.20 ± 0.01) with age (P < 0.01). No significant differences in aggrecan and Type II collagen content of L1-L6 intervertebral discs in Group A were noted (P > 0.05). Mean ADC values of different intervertebral regions were positively correlated with aggrecan and Type II collagen content (aggrecan: r = 0.631, P < 0.01; Type II collagen: r = 0.680, P < 0.01). CONCLUSION: 7.0T MRI-DWI could be applied to effectively diagnose and research early IDD in tiny variations.

Isolation and prolonged expansion of oral mesenchymal stem cells under clinical-grade, GMP-compliant conditions differentially affects "stemness" properties.

BACKGROUND: Development of clinical-grade cell preparations is central to meeting the regulatory requirements for cellular therapies under good manufacturing practice-compliant (cGMP) conditions. Since addition of animal serum in culture media may compromise safe and efficient expansion of mesenchymal stem cells (MSCs) for clinical use, this study aimed to investigate the potential of two serum/xeno-free, cGMP culture systems to maintain long-term "stemness" of oral MSCs (dental pulp stem cells (DPSCs) and alveolar bone marrow MSCs (aBMMSCs)), compared to conventional serum-based expansion. METHODS: DPSC and aBMMSC cultures (n = 6/cell type) were established from pulp and alveolar osseous biopsies respectively. Three culture systems were used: StemPro_MSC/SFM_XenoFree (Life Technologies); StemMacs_MSC/XF (Miltenyi Biotek); and α-MEM (Life Technologies) with 15% fetal bovine serum. Growth (population doublings (PDs)), immunophenotypic (flow cytometric analysis of MSC markers) and senescence (ß-galactosidase (SA-ß-gal) activity; telomere length) characteristics were determined during prolonged expansion. Gene expression patterns of osteogenic (ALP, BMP-2), adipogenic (LPL, PPAR-γ) and chondrogenic (ACAN, SOX-9) markers and maintenance of multilineage differentiation potential were determined by real-time PCR. RESULTS: Similar isolation efficiency and stable growth dynamics up to passage 10 were observed for DPSCs under all expansion conditions. aBMMSCs showed lower cumulative PDs compared to DPSCs, and when StemMacs was used substantial delays in cell proliferation were noted after passages 6-7. Serum/xeno-free expansion produced cultures with homogeneous spindle-shaped phenotypes, while serum-based expansion preserved differential heterogeneous characteristics of each MSC population. Prolonged expansion of both MSC types but in particular the serum/xeno-free-expanded aBMMSCs was associated with downregulation of CD146, CD105, Stro-1, SSEA-1 and SSEA-4, but not CD90, CD73 and CD49f, in parallel with an increase of SA-gal-positive cells, cell size and granularity and a decrease in telomere length. Expansion under both serum-free systems resulted in "osteogenic pre-disposition", evidenced by upregulation of osteogenic markers and elimination of chondrogenic and adipogenic markers, while serum-based expansion produced only minor changes. DPSCs retained a diminishing (CCM, StemPro) or increasing (StemMacs) mineralization potential with passaging, while aBMMSCs lost this potential after passages 6-7 under all expansion conditions. CONCLUSIONS: These findings indicate there is still a vacant role for development of qualified protocols for clinical-grade expansion of oral MSCs; a key milestone achievement for translation of research from the bench to clinics.

Wide bandwidth nanomechanical assessment of murine cartilage reveals protection of aggrecan knock-in mice from joint-overuse.

Aggrecan loss in human and animal cartilage precedes clinical symptoms of osteoarthritis, suggesting that aggrecan loss is an initiating step in cartilage pathology. Characterizing early stages of cartilage degeneration caused by aging and overuse is important in the search for therapeutics. In this study, atomic force microscopy (AFM)-based force-displacement micromechanics, AFM-based wide bandwidth nanomechanics (nanodynamic), and histologic assessments were used to study changes in distal femur cartilage of wildtype mice and mice in which the aggrecan interglobular domain was mutated to make the cartilage aggrecanase-resistant. Half the animals were subjected to voluntary running-wheel exercise of varying durations. Wildtype mice at three selected age groups were compared. While histological assessment was not sensitive enough to capture any statistically significant changes in these relatively young populations of mice, micromechanical assessment captured changes in the quasi-equilibrium structural-elastic behavior of the cartilage matrix. Additionally, nanodynamic assessment captured changes in the fluid-solid poroelastic behavior and the high frequency stiffness of the tissue, which proved to be the most sensitive assessment of changes in cartilage associated with aging and joint-overuse. In wildtype mice, aging caused softening of the cartilage tissue at the microscale and at the nanoscale. Softening with increased animal age was found at high loading rates (frequencies), suggesting an increase in hydraulic permeability, with implications for loss of function pertinent to running and impact-injury. Running caused substantial changes in fluid-solid interactions in aggrecanase-resistant mice, suggestive of tissue degradation. However, higher nanodynamic stiffness magnitude and lower hydraulic permeability was observed in running aggrecanase-resistant mice compared to running wildtype controls at the same age, thereby suggesting protection from joint-overuse.

Study design: in vitro and in vivo assessment of bone morphogenic protein 2 combined with platelet-rich plasma on treatment of disc degeneration.

OBJECTIVE: Our aim was to investigate the biological effects of bone morphogenic protein 2 (BMP2) on the differentiation of bone marrow mesenchymal stem cells (BMSCs) into chondrocyte-like cells in platelet-rich plasma (PRP) gel in vitro. In addition, the effectiveness of BMP2-transduced BMSCs in combined with PRP gel to repair the degenerated intervertebral disc in a rabbit model was also evaluated. Previous studies have shown that tissue engineering provides many promising advantages to treating disc degeneration and may reverse the pathological process of disc degeneration. METHODS: The expressions of types I, II and X collagen, aggrecan and Sox9 of the BMP2-transduced BMSCs in monolayer or PRP gel were examined by reverse transcriptase polymerase chain reaction (RT-PCR). Sixty New Zealand white rabbits were divided into five groups: 12 normal controls; 12 puncture operated with only disc degeneration being induced; 12 PRP transplantation animals; 12 BMSC and PRP-transplantation animals; 12 BMP2-transduced BMSCs and PRP-transplantation animals. The effect of BMP2-transduced BMSCs on degenerated discs were evaluated by magnetic resonance image (MRI) scan, histology, immunohistochemistry and Western blot analysis. RESULTS: BMP2 could facilitate chondrogenic differentiation of BMSCs in monolayer or PRP gel. The discs treated with BMP2-transduced BMSCs exhibited relatively well-preserved nucleus pulposus (NP) structure. Significantly higher T2-weighted signal intensity and a greater amount of extracellular matrix were observed in the BMP2-transduced BMSC group compared with other groups. In addition, the presences of BMP2-transduced BMSCs were identified at week 12 postoperatively in vivo. CONCLUSIONS: BMP2-transduced BMSCs can maintain the chondrocyte-like phenotype in PRP gel in vitro, and the combined use of these two agents can significantly promote repair of the degenerated discs in vivo.

A short-term pharmacodynamic model for monitoring aggrecanase activity: injection of monosodium iodoacetate (MIA) in rats and assessment of aggrecan neoepitope release in synovial fluid using novel ELISAs.

OBJECTIVE: To develop a short-term in vivo model in rats, with an enzyme-linked immunosorbent assay (ELISA) readout for specific aggrecanase-cleaved aggrecan fragments, to facilitate testing of aggrecanase inhibitors. METHODS: Monosodium iodoacetate (MIA), a metabolic inhibitor, was injected into the right knee joint of male Lewis rats and the release of aggrecanase-cleaved fragments of aggrecan containing the NITEGE or ARGN neoepitope was measured in the synovial fluid at 7 days post MIA injection using novel ELISAs. The ELISAs utilize a commercial antibody directed against the hyaluronic-acid binding region (HABR) of aggrecan, in combination with either an alpha-NITEGE antibody (NITEGE ELISA) or an alpha-ARGS/BC3 antibody (ARGS ELISA), to detect aggrecanase-cleavage of aggrecan within the interglobular domain (IGD). Aggrecan fragments present in in vitro digests, in cytokine-treated cartilage explant culture supernatants and in rat synovial fluid lavage samples were detected and quantified using the two ELISAs. Small molecule inhibitors of aggrecanase activity were dosed orally on days 3-7 to determine their ability to inhibit MIA-induced generation of the NITEGE and ARGN neoepitopes measured in the rat synovial fluid. RESULTS: The NITEGE assay was shown to specifically detect the N-terminal fragment of aggrecan comprising the G1 domain and the NITEGE neoepitope sequence. This assay can readily measure aggrecanase-cleaved bovine, human and rat aggrecan without the need for deglycosylation. The ARGS assay specifically detects C-terminal fragments of aggrecan comprising the ARGS/ARGN neoepitope and the G2 domain. Keratan sulfate (KS) residues of aggrecan interfere with this ELISA, and hence this assay works well with native rat articular cartilage aggrecan (that lacks KS residues) and with deglycosylated bovine and human aggrecan. Injection of MIA into the rat knee joints resulted in a time-dependent increase in the release of aggrecanase-cleaved aggrecan fragments into the synovial fluid and treatment with an aggrecanase inhibitor resulted in a dose-dependent inhibition of the generation of these neoepitopes. CONCLUSIONS: We have established a short-term in vivo model in rats that involves measurement of synovial fluid biomarkers that are dependent on aggrecanase activity in the joint. The short duration of the model combined with the mechanistic biomarker readout makes it very useful for the initial in vivo screening of aggrecanase inhibitors prior to testing them in time and resource-intensive disease models of osteoarthritis (OA).

Assessment of the catabolic effects of interleukin-1beta on proteoglycan metabolism in equine cartilage cocultured with synoviocytes.

OBJECTIVE: To evaluate the effects of interleukin (IL)-1beta on proteoglycan metabolism in equine cartilage explants when cultured in the presence of synoviocytes. SAMPLE POPULATION: Samples of cartilage and synovium collected from the femoropatellar joints of three 2- to 3-year-old horses. PROCEDURES: 3 experimental groups were established: cartilage explants only, synoviocytes only, and cartilage explants-synoviocytes in coculture. In each group, samples were cultured with or without IL-1beta (10 ng/mL) for 96 hours. Glycosaminoglycan (GAG) content of cartilage and medium samples was measured by use of a spectrophotometric assay; RNA was isolated from synoviocytes and cartilage and analyzed for expression of matrix metalloproteinases (MMP)-3 and -13 (cartilage and synoviocytes), aggrecan (cartilage), collagen type IIB (cartilage), and 18S as a control (cartilage and synoviocytes) by use of quantitative PCR assays. Cartilage matrix metachromasia was assessed histochemically. RESULTS: IL-1beta-induced GAG loss from cartilage was significantly less in cocultures than in cartilage-only cultures. Cartilage aggrecan gene expression was also significantly less downregulated and synoviocyte MMP-3 expression was less upregulated by IL-1beta in cocultures, compared with cartilage- and synoviocyte only cultures. Histochemical findings supported the molecular and biochemical results and revealed maintenance of matrix metachromasia in cocultured cartilage treated with IL-1beta. CONCLUSIONS AND CLINICAL RELEVANCE: Results suggest that synoviocytes secrete 1 or more mediators that preferentially protect matrix GAG metabolism from the degradative effects of IL-1beta. Further studies involving proteomic and microarray approaches in similar coculture systems may elucidate novel therapeutic targets for the treatment of osteoarthritis.