Cartilage disease is a leading cause of lameness and loss of function in animals and disability in humans. The limited potential for chondrocyte proliferation and the avascular nature of cartilage tissue contribute to the poor healing potential of adult cartilage, eventually leading to osteoarthritis.1 Tissue engineering offers new strategies for treating damaged or diseased cartilage. In this process, cells are allowed to proliferate and organize their extracellular matrix in a 3-D lattice to form ex vivo a clinically functional tissue with histochemical, biochemical, and biomechanical properties identical to those of native, healthy tissue. The most common approach for the restoration of cartilage surfaces consists of transplanting autologous chondrocytes harvested from a non-weight-bearing region and expanded in vitro.2,3 However, this technique requires 2 surgeries, and its application is further limited by damage caused at the donor site.
The use of MSCs for the treatment of joint disease has recently generated a great deal of interest because these cells are easier to harvest from a patient than chondrocytes, are immunoprivileged, and can differentiate into several cell types. Perhaps as important, they are able to regenerate the underlying subchondral bone as well as the damaged cartilage,4,5 which expands the repertoire of MSCs to include repair of osteochondral defects. In contrast to chondrocytes, MSCs integrate better into damaged sites6,7 and make repairs that may last longer than those in which chondrocytes are used.8 Nevertheless, MSCs injected into a fibrin vehicle reportedly fail to improve the long-term histologic appearance or biochemical composition of full-thickness cartilage lesions in horses.9 These findings may reflect deficient retention of MSCs after injection into the lesion or an inability of MSCs to achieve the genetic profile of a fully differentiated chondrocyte. Research efforts are focused on improving the chondrogenic potential of MSCs in vitro as well as their delivery in vivo.
Techniques based on needle reinfusion of a cell suspension into a cartilage defect carry the risk of rapid dissemination of cells away from the lesion.10 Delivering MSCs cultured on biodegradable carriers, however, improves their retention within the damaged area and provides local biomechanical support. In addition, recent findings support the use of scaffolds as an additional strategy to modulate the differentiation of MSCs, in concert with growth factors and cytokines.11,12 In that context, chitosan and HA are attractive candidates for joint resurfacing because of their excellent biocom-patibility and similarity of structure with natural constituents of articular cartilage.
Chitosan is formed by alkaline deacetylation of chitin, the second most abundant natural polysaccha-ride, primarily obtained as a natural subproduct of shellfish.13 Its superior biocompatibility has been attributed to its structural similarity with GAGs, which are naturally present in the ECM of cartilage.14 Chitosan has received FDA approval for use in humans in the treatment of wounds and has recently attracted a great deal of interest as a drug delivery system, bone graft substitute, and cartilage engineering substrate.15–17 Macroporous chitosan sponges promote matrix production by individual cells and, compared with polyglycolic acid fibers, preserve the phenotypic appearance of chondrocytes.18 Chondrogenesis by MSCs on a chitosan copolymer gel is also possible.19
Hyaluronic acid has been a mainstay of the clinical arsenal against joint disease in the horse for decades. Intra-articular injection of HA in equids was first reported in 1970,20 and its use as a sole treatment, resulting in lameness reduction, has been supported by the findings of several clinical studies.21,22 Experimentally, intra-articular injection of HA has been shown to improve weight bearing after radiocarpal bone osteochondral fracture as determined by force plate analysis and to preserve articular cartilage integrity as determined by histologic analysis.23–25 Results of clinical studies in horses26 and humans27 indicate that high-MW HA has better clinical efficacy than low-MW HA. The improved therapeutic effects associated with the high-MW compound may be attributed to its anti-inflammatory effects, including inhibition of phagocytosis, chemotaxis of inflammatory cells, and synthesis of prostaglandin as well as removal of free radicals and other reactive oxygen species.28
In vitro, HA reportedly prevents interleuken-1-induced proteoglycan release from cultured canine chondrocytes and inhibits prostaglandin E2 synthesis.29 In vivo, high-MW HA blocks the expression of inflammatory cytokines and matrix metalloproteinase-3 in arthritic joints.30 High-MW HA appears to exert its anti-inflammatory effects by binding firmly with HA receptors on synovial cells, inhibiting activation of p38 mitogen-activated protein kinase, and blocking transcription of the cycloxygenase-2 gene.30 The actions of HA appear partially mediated by activation of receptor CD44, intercellular adhesion molecule 1, and the receptor for hyaluronan-mediated motility.31 Hyaluronic acid also promotes CD44-mediated, dose-dependent recruitment of MSCs.32 These data provide the rationale for incorporating HA into the scaffold rather than the culture medium as an alternative strategy to enhance MSC chondrogenesis while improving cell attachment and dispersal into the scaffold. The potential for synergetic effects of HA incorporation into the scaffold and supplementation of the medium has not been addressed to our knowledge.
The purpose of the study reported here was to establish the dose-dependent effects of high-MW HA supplementation on matrix production and chondrogenic differentiation of MSCs cultured on chitosan sponges. A second objective was to determine the extent to which MSC matrix production and chondrogenesis could be influenced by incorporation of high-MW HA into chitosan scaffolds. Our hypothesis was that high-MW HA added directly into the culture medium or incorporated into 3-D hybrid chitosan-HA sponge scaffolds would stimulate chondrogenesis by MSCs cultured in these scaffolds.
Materials and Methods
MSCs—A mesenchymal cell linea derived from a multipotent mouse bone marrow stromal precursor was selected on the basis of previous evidence of its differentiation into chondrocytes.33 Cells were expanded as recommended by the supplier. Complete expansion medium consisted of DMEM,b 1% penicillin (100 U/mL) and streptomycin (100 μg/mL),c and 10% fetal bovine serum.b Passage 3 cells were expanded once in a 37°C, 5% CO2 incubator to 90% confluence, washed, and counted by use of trypan blue stain and resuspended in a defined chondrogenic medium consisting of DMEM-high glucoseb with a universal culture supplementd (insulin [6.25 μg/mL], transferrin [6.25 μg/mL], selenous acid [6.25 μg/mL], and linoleic acid [5.33 μg/mL]), bovine serum albumin (1.25 μg/mL), pyruvate (1 mM), ascorbate 2-phosphate (37.5 μg/mL), TGF-β1 (10 ng/mL),e and 1% penicillin-streptomycin.34
Scaffolds—Chitosan (0% HA) and hybrid chitosan-HA (0.01% HA) scaffolds were prepared by means of a freeze-drying technique.18,35 Briefly, 2% (wt/vol) chitosan fakesf and, for hybrid chitosan-HA scaffolds, 0.01% sodium hyaluronateg were dissolved in 0.2M acetic acid and stirred for 48 hours. The solution was poured into a 96-well mold, cooled at a controlled rate of −0.3°C for 72 hours, and lyophilized for 48 hours to produce macroporous sponges with a diameter of 5 mm and a height of 3 mm.35 Prior to MSC seeding, all scaffolds were rehydrated through a series of ethanol and PBS solutions and incubated in complete expansion medium at 37°C for 24 hours on a shaker incubator.36 The microstructure of scaffolds was verified via SEM.
Seeding—Each scaffold was placed in a well of a 24-well non-tissue culture-treated plate. An MSC suspension (20 μL at 1 × 108 cells/mL [ie, 2 × 106 cells/scaffold]) was pipetted onto each scaffold and allowed to incubate at 37°C in a 5% CO2 incubator for 1 hour. Chondrogenic medium (2 mL) was added to each well and placed on a platform shaker oscillating at 25 rpm. Chondrogenic medium consisted of the defined chondrogenic medium plus 100nM dexamethasone, tapered to 0nM starting on day 6.37 All constructs were maintained for 48 hours or 21 days in a 37°C, 5% CO2 incubator with medium changes every 2 to 3 days.
In vitro chondrogenesis—Eight treatment groups of MSC-scaffold constructs were compared, each with 1 of 4 HA concentrations in the medium and with 1 of 2 scaffold types. Hyaluronic acidg was added to the medium in 4 concentrations: 0, 0.5, 1, and 2 mg/mL. Thus, the following treatment groups were established: 0% scaffold HA content and medium HA concentrations of 0, 0.5, 1.0, and 2.0 mg/mL or 0.01% scaffold HA content and medium HA concentrations of 0, 0.5, 1.0, and 2.0 mg/mL.
The 0.5 mg/mL dose of HA reportedly increases proliferation and matrix production by chondrocytes cultured in gelatin sponges.38 The highest dose (2 mg/mL) approximates the concentration achieved in the joint after clinical intra-articular injection of HA in horses.39 The combination of no HA in the medium, the scaffold, or both was included to verify the lack of interference between HA supplementation and the assay. The entire experiment was repeated in triplicate.
Cell viability—Cell viability was evaluated for each treatment group 48 hours after seeding to confirm the high viability of cells within the constructs. This evaluation was not repeated 21 days after seeding because the presence of extracellular matrix would interfere with the results. Constructs were sectioned into 1.5-mm-thick slices prior to staining by use of a commercial viability-cytotoxicity kit.h Fluorescent confocal microscopyi with argon and krypton lasers at excitation wavelengths of 488 and 568 nm allowed the visualization of calcein acetoxymethyl ester, which labels live cells with green fluorescence, and ethidium homodimer-1, which labels dead cells with red fluorescence. Constructs were washed 3 times in sterile PBS solution for 2 minutes and sectioned perpendicularly into 1.5-mm-thick slices by use of a single-edged razor blade. Slices from the periphery and center of each construct were placed on a glass slide and immersed in 200 μL of PBS solution containing 2mM calcein acetoxymethyl ester and 4mM ethidium homodimer-1 prior to incubation for 40 minutes at room temperature (approx 22°C). The intensities of viable and nonviable cells were measured from 4 sites/construct at a magnification of 40× and from 2 sites at 10×. The images were analyzed by use of computer softwarej to determine the percentage of viable cells. Cell viability was measured as the relative intensity of each fluorescent dye on at least 3 fields of view/section within each construct and expressed as percentage of live cells.
DNA quantification—Content of DNA was evaluated in each treatment group 48 hours and 21 days after seeding. Constructs (scaffold seeded with MSCs) were weighed before and after dehydration to determine water content and dry weight. They were then digested in papainc according to their dry weight for 16 hours at 60°C and assayed with a fluorometric assay.k The amount of DNA was determined by use of a microplate spectrophotometer,1 with excitation at 348 nm and emission at 456 nm.
GAG and collagen II content—The quality of the engineered tissue composition was evaluated 21 days after seeding by examining GAG and collagen II. Quantification of GAG was performed on the same 21-day, papain-digested samples used for DNA quantification. Glycosaminoglycan content was determined by use of a GAG quantification assaym in which 1,9-dimethylmethylene blue chloride was used. Dye absorption was measured at 530 nm on a microplate spectrophotometer.l Shark chondroitin sulfatec (5 to 50 μg/mL) was used as a standard. The GAG content was normalized to DNA content of the corresponding construct to account for differences in cell numbers.
Collagen type II content was determined by means of an ELISA that involved modification of a commercial type II collagen detection kit,n as described elsewhere.35 Briefly, constructs were digested 4 times in pepsinc for 24 hours each, followed by a 24-hour elastasec digestion. The supernatant was recovered and pooled after each digestion. The pH was neutralized with 1M Tris buffer. The ELISA was performed following the manufacturer's protocol. Collagen II was measured on a microplate spectro-photometerk at 490 nm. Collagen II content was normalized to the mean DNA content for each treatment group to account for differences in cell numbers.
SEM—Cell attachment and distribution were evaluated for each treatment group via SEM 48 hours after seeding. The presence of extracellular matrix within the scaffolds hindered evaluation of cell attachment and distribution 21 days after seeding. Constructs were bisected prior to SEM to evaluate the periphery and the central section of each sample. They were prepared following a protocol previously published by our group for the evaluation of constructs.18,35 Briefly, constructs were fixed in a 2.5% gluteraldehyde solution with sodium cacodylate buffer for 2 hours, stained with 1% osmium tetroxide in 0.1M sodium cacodylate for 90 minutes, dehydrated through an ethanol series, and placed in hexamethyldisilazane under a fume hood until completely dry. Constructs were mounted and sputter coated with gold-palladium prior to examination with an SEM at 5kV.° Criteria evaluated included cell distribution, morphological characteristics, size, and attachment and presence of cytoplasmic extensions. The overall cell coverage on the scaffolds was estimated on each of 10 images (800× magnification) obtained at random sites (5 from the center and 5 from the periphery). A grid was overlaid on each SEM image to determine cell coverage. The number of grid squares completely filled with cells was counted. In addition, the portion covered in partially filled squares was estimated, summed, and added to the number of filled squares. The number of filled squares was then converted to a percentage of the scaffold surface covered by cells in each image.
Histologic evaluation—Sections of engineered tissue were examined for scaffold morphological characteristics, cell distribution, and the presence, distribution, and relative abundance of pericellular proteoglycans 21 days after seeding. Constructs were fixed in 4% paraformaldehyde for 2 hours, embedded in paraffin, and cut via microtome to produce 1-μm-thick sections. A minimum of 3 sections/sample were stained with 1 of 3 staining protocols: H&E, safranin-O and fast green (for proteoglycans), and anti-aggrecan antibody.p The area staining positive for proteoglycans or aggrecan was measured by use of computer softwareq and expressed as a percentage of the total area covered by the construct in each image. All images obtained at a magnification of 40× (a minimum of 3 images from each of 3 samples/treatment) were included in the analysis.
Statistical analysis—The live-to-dead cell ratio and DNA, GAG, and collagen II contents of the constructs were compared between scaffold types and among medium HA concentrations. The potential interaction between scaffold type and media HA concentration was also analyzed. In addition to analyzing the data for each of the 8 treatment groups, data was pooled by scaffold type (2 groups: chitosan vs hybrid chitosan-HA) and by medium (4 groups based on HA concentrations in the media). Hypothesis testing was performed by use of a generalized linear mixed model as constructed with a software program.r The histologic data were analyzed with the Kruskal-Wallis test and different computer software.s A value of P < 0.05 was considered significant for all analyses.
Results
Cell viability—Forty-eight hours after seeding on chitosan (0% HA) or hybrid chitosan-HA (0.01% HA) scaffolds, the proportion of viable murine bone marrow MSCs was evaluated for each of the 8 treatment groups, each of which varied in HA concentration. The viability of cells was similar among groups, with a mean of 79.0 ± 15.6% cells staining as viable in the constructs (data not shown).
DNA quantification—Content of DNA was evaluated in each treatment group 48 hours and 21 days after seeding to obtain an approximation of cell numbers on each scaffold and to use these data for normalization of ECM protein production. There was no significant difference in DNA quantity among the 8 treatment groups at either the 48-hour or 21-day extraction times (data not shown).
GAG and collagen II content—The engineered tissue composition was evaluated on the basis of GAG and collagen II content 21 days after seeding. No GAG was detected in the blank scaffolds, and GAG content significantly (P < 0.001) increased with increasing HA concentration in the culture medium (Figure 1). The addition of HA to the chitosan to create hybrid scaffolds appeared to have little influence on GAG content, and there was no difference between constructs with chitosan and hybrid scaffolds.
Mean ± SE GAG content normalized to DNA content of murine bone marrow-derived MSC-chitosan scaffold constructs after 21 days of incubation in 8 treatment conditions. The 8 treatment conditions consisted of 0% (S 0) or 0.01% (S 0.01) scaffold HA content (wt/vol) and culture medium HA concentrations of 0 mg/mL (M 0), 0.5 mg/mL (M 0.5), 1.0 mg/mL (M 1.0), and 2.0 mg/mL (M 2.0). Different letters (A-D) above bars indicate significant (P < 0.05) differences among the various culture medium HA concentrations.
Citation: American Journal of Veterinary Research 72, 1; 10.2460/ajvr.72.1.42
Mean ± SE GAG content normalized to DNA content of murine bone marrow-derived MSC-chitosan scaffold constructs after 21 days of incubation in 8 treatment conditions. The 8 treatment conditions consisted of 0% (S 0) or 0.01% (S 0.01) scaffold HA content (wt/vol) and culture medium HA concentrations of 0 mg/mL (M 0), 0.5 mg/mL (M 0.5), 1.0 mg/mL (M 1.0), and 2.0 mg/mL (M 2.0). Different letters (A-D) above bars indicate significant (P < 0.05) differences among the various culture medium HA concentrations.
Citation: American Journal of Veterinary Research 72, 1; 10.2460/ajvr.72.1.42
Mean ± SE GAG content normalized to DNA content of murine bone marrow-derived MSC-chitosan scaffold constructs after 21 days of incubation in 8 treatment conditions. The 8 treatment conditions consisted of 0% (S 0) or 0.01% (S 0.01) scaffold HA content (wt/vol) and culture medium HA concentrations of 0 mg/mL (M 0), 0.5 mg/mL (M 0.5), 1.0 mg/mL (M 1.0), and 2.0 mg/mL (M 2.0). Different letters (A-D) above bars indicate significant (P < 0.05) differences among the various culture medium HA concentrations.
Citation: American Journal of Veterinary Research 72, 1; 10.2460/ajvr.72.1.42
Similar results were found for type II collagen production. There was a significant (P < 0.001) effect of increasing HA concentration in the culture medium, by which a greater HA concentration yielded greater collagen type II content (Figure 2). The scaffold type (chitosan vs hybrid) did not significantly affect collagen II content. Thus, both GAG and collagen II production increased in a dose-dependent manner with increasing concentrations of HA in the culture medium.
Mean ± SE collagen type II content normalized to DNA content of murine bone marrow-derived MSC-chitosan scaffold constructs after 21 days of incubation in 8 treatment conditions. See Figure 1 for key.
Citation: American Journal of Veterinary Research 72, 1; 10.2460/ajvr.72.1.42
Mean ± SE collagen type II content normalized to DNA content of murine bone marrow-derived MSC-chitosan scaffold constructs after 21 days of incubation in 8 treatment conditions. See Figure 1 for key.
Citation: American Journal of Veterinary Research 72, 1; 10.2460/ajvr.72.1.42
Mean ± SE collagen type II content normalized to DNA content of murine bone marrow-derived MSC-chitosan scaffold constructs after 21 days of incubation in 8 treatment conditions. See Figure 1 for key.
Citation: American Journal of Veterinary Research 72, 1; 10.2460/ajvr.72.1.42
SEM—The microstructure of pure chitosan differed from that of hybrid chitosan-HA scaffolds when examined via SEM 48 hours after cell seeding. The chitosan-HA scaffolds contained small extensions and fakes that were not evident on scaffolds lacking HA (Figure 3). All MSC-scaffold constructs contained cells primarily with the size and stellate morphology consistent with MSCs, whereas other cells appeared to be undergoing chondrogenic differentiation given their size and rounded appearance. The cells generally appeared in clumps congregated at or near the scaffold surface with declining numbers toward the center of constructs (Figure 4). At 48 hours, there was a significant (P < 0.001) difference in cell coverage of the scaffolds (as judged via SEM) among the constructs incubated in media differing in HA concentration. Constructs cultured in medium supplemented with 0.5 mg/mL HA had the greatest cellular coverage (Figure 5).
Scanning electron microscopy photomicrographs of murine bone marrow-derived MSC-chitosan scaffold constructs after 48 hours of incubation in various treatment conditions. A—Internal structure of a chitosan scaffold with 0% HA, incubated in culture medium without HA. B—Internal structure of a chitosan scaffold containing 0.01% HA (hybrid chitosan-HA scaffold) incubated in culture medium with an HA concentration of 1. 0 mg/mL. Notice the small extensions (arrows) and HA fakes (arrowhead) seen only on the hybrid scaffolds. The inset shows magnification of small extensions in a hybrid scaffold incubated with less HA (0.5 mg/mL). C—Typical MSCs with a characteristic fat, stellate appearance. D—Rounded cells (arrow) are bone marrow MSCs differentiating into chondrocytes. Bar = 10 μm in all images.
Citation: American Journal of Veterinary Research 72, 1; 10.2460/ajvr.72.1.42
Scanning electron microscopy photomicrographs of murine bone marrow-derived MSC-chitosan scaffold constructs after 48 hours of incubation in various treatment conditions. A—Internal structure of a chitosan scaffold with 0% HA, incubated in culture medium without HA. B—Internal structure of a chitosan scaffold containing 0.01% HA (hybrid chitosan-HA scaffold) incubated in culture medium with an HA concentration of 1. 0 mg/mL. Notice the small extensions (arrows) and HA fakes (arrowhead) seen only on the hybrid scaffolds. The inset shows magnification of small extensions in a hybrid scaffold incubated with less HA (0.5 mg/mL). C—Typical MSCs with a characteristic fat, stellate appearance. D—Rounded cells (arrow) are bone marrow MSCs differentiating into chondrocytes. Bar = 10 μm in all images.
Citation: American Journal of Veterinary Research 72, 1; 10.2460/ajvr.72.1.42
Scanning electron microscopy photomicrographs of murine bone marrow-derived MSC-chitosan scaffold constructs after 48 hours of incubation in various treatment conditions. A—Internal structure of a chitosan scaffold with 0% HA, incubated in culture medium without HA. B—Internal structure of a chitosan scaffold containing 0.01% HA (hybrid chitosan-HA scaffold) incubated in culture medium with an HA concentration of 1. 0 mg/mL. Notice the small extensions (arrows) and HA fakes (arrowhead) seen only on the hybrid scaffolds. The inset shows magnification of small extensions in a hybrid scaffold incubated with less HA (0.5 mg/mL). C—Typical MSCs with a characteristic fat, stellate appearance. D—Rounded cells (arrow) are bone marrow MSCs differentiating into chondrocytes. Bar = 10 μm in all images.
Citation: American Journal of Veterinary Research 72, 1; 10.2460/ajvr.72.1.42
Photomicrographs showing the distribution of murine bone marrow-derived MSCs on chitosan scaffolds as seen via SEM. Cell numbers gradually decline and spacing increases the further from the scaffold surface they are. A—Surface of a chitosan scaffold containing 0.01% HA incubated with MSCs in culture medium containing no HA. A heavy coating of cells is evident. B—Interior of the same scaffold showing clumps of cells. C—Deep within a chitosan scaffold containing no HA and incubated with MSCs in culture medium containing no HA. A few scattered cells are evident. Bar = 10 μm in all images.
Citation: American Journal of Veterinary Research 72, 1; 10.2460/ajvr.72.1.42
Photomicrographs showing the distribution of murine bone marrow-derived MSCs on chitosan scaffolds as seen via SEM. Cell numbers gradually decline and spacing increases the further from the scaffold surface they are. A—Surface of a chitosan scaffold containing 0.01% HA incubated with MSCs in culture medium containing no HA. A heavy coating of cells is evident. B—Interior of the same scaffold showing clumps of cells. C—Deep within a chitosan scaffold containing no HA and incubated with MSCs in culture medium containing no HA. A few scattered cells are evident. Bar = 10 μm in all images.
Citation: American Journal of Veterinary Research 72, 1; 10.2460/ajvr.72.1.42
Photomicrographs showing the distribution of murine bone marrow-derived MSCs on chitosan scaffolds as seen via SEM. Cell numbers gradually decline and spacing increases the further from the scaffold surface they are. A—Surface of a chitosan scaffold containing 0.01% HA incubated with MSCs in culture medium containing no HA. A heavy coating of cells is evident. B—Interior of the same scaffold showing clumps of cells. C—Deep within a chitosan scaffold containing no HA and incubated with MSCs in culture medium containing no HA. A few scattered cells are evident. Bar = 10 μm in all images.
Citation: American Journal of Veterinary Research 72, 1; 10.2460/ajvr.72.1.42
Mean ± SE percentages of murine bone marrow- derived MSC coverage of chitosan scaffolds after incubation for 48 hours in 8 treatment conditions. See Figure 1 for key.
Citation: American Journal of Veterinary Research 72, 1; 10.2460/ajvr.72.1.42
Mean ± SE percentages of murine bone marrow- derived MSC coverage of chitosan scaffolds after incubation for 48 hours in 8 treatment conditions. See Figure 1 for key.
Citation: American Journal of Veterinary Research 72, 1; 10.2460/ajvr.72.1.42
Mean ± SE percentages of murine bone marrow- derived MSC coverage of chitosan scaffolds after incubation for 48 hours in 8 treatment conditions. See Figure 1 for key.
Citation: American Journal of Veterinary Research 72, 1; 10.2460/ajvr.72.1.42
Histologic evaluation—After 21 days of incubation, all stained sections of the constructs had a similar histologic appearance. The constructs contained discrete areas in which cells were embedded in matrix, particularly at or near the periphery (Figure 6). Although not statistically assessed, cell numbers and penetration within scaffolds appeared to improve in constructs cultured in medium with higher HA concentrations (data not shown). Images of constructs stained for proteoglycan suggested a correlation between matrix production and medium supplementation with increasing HA concentrations (Figures 7 and 8). The size of areas staining for proteoglycan and aggrecan confirmed the results of the biochemical analyses, as both increased with increasing HA concentration in the medium (P = 0.011 and P < 0.001, respectively; Figure 9). There was no difference between constructs with different scaffold types cultured in the same medium. When normalized to the amount of DNA (group means), the significance of HA concentration in the culture medium increased to a value of P < 0.001 for proteoglycans. Interestingly, upon DNA-based normalization, hybrid chitosan-HA constructs contained more matrix than pure chitosan constructs (aggrecan, P = 0.002; proteoglycan, P = 0.021; data not shown).
Photomicrograph of a murine bone marrow-derived MSC-chitosan scaffold without HA (red), incubated for 3 weeks in culture medium containing 2 mg of HA/mL and stained to visualize cell (pink with purple nuclei) distribution. The upper surface of the construct is in the upper right corner of the image; deepest portion is at the lower left corner. H&E stain; bar = 50 μm.
Citation: American Journal of Veterinary Research 72, 1; 10.2460/ajvr.72.1.42
Photomicrograph of a murine bone marrow-derived MSC-chitosan scaffold without HA (red), incubated for 3 weeks in culture medium containing 2 mg of HA/mL and stained to visualize cell (pink with purple nuclei) distribution. The upper surface of the construct is in the upper right corner of the image; deepest portion is at the lower left corner. H&E stain; bar = 50 μm.
Citation: American Journal of Veterinary Research 72, 1; 10.2460/ajvr.72.1.42
Photomicrograph of a murine bone marrow-derived MSC-chitosan scaffold without HA (red), incubated for 3 weeks in culture medium containing 2 mg of HA/mL and stained to visualize cell (pink with purple nuclei) distribution. The upper surface of the construct is in the upper right corner of the image; deepest portion is at the lower left corner. H&E stain; bar = 50 μm.
Citation: American Journal of Veterinary Research 72, 1; 10.2460/ajvr.72.1.42
Representative photomicrographs of extracellular matrix proteins on murine bone marrow-derived MSC-chitosan scaffold constructs (without HA) incubated in culture medium containing various concentrations of HA for 48 hours or 3 weeks in various conditions and stained for proteoglycans. Scaffolds appear dark, and proteoglycans appear as teal-colored strands. A—Culture medium contained no HA. B—Culture medium contained HA at 0.05 mg/mL. C—Culture medium contained HA at 1.0 mg/mL. D—Culture medium contained HA at 2.0 mg/mL. E—Constructs incubated for 21 days in growth medium without HA. There are no extracellular fibers present. F—Constructs incubated for 21 days in chondrogenic medium containing HA (1.0 mg/mL). Notice the extracellular fibers staining for proteoglycans. Saffranin-O and fast green stain; bar = 50 μm in panels A through D and 10 μm in panels E and F.
Citation: American Journal of Veterinary Research 72, 1; 10.2460/ajvr.72.1.42
Representative photomicrographs of extracellular matrix proteins on murine bone marrow-derived MSC-chitosan scaffold constructs (without HA) incubated in culture medium containing various concentrations of HA for 48 hours or 3 weeks in various conditions and stained for proteoglycans. Scaffolds appear dark, and proteoglycans appear as teal-colored strands. A—Culture medium contained no HA. B—Culture medium contained HA at 0.05 mg/mL. C—Culture medium contained HA at 1.0 mg/mL. D—Culture medium contained HA at 2.0 mg/mL. E—Constructs incubated for 21 days in growth medium without HA. There are no extracellular fibers present. F—Constructs incubated for 21 days in chondrogenic medium containing HA (1.0 mg/mL). Notice the extracellular fibers staining for proteoglycans. Saffranin-O and fast green stain; bar = 50 μm in panels A through D and 10 μm in panels E and F.
Citation: American Journal of Veterinary Research 72, 1; 10.2460/ajvr.72.1.42
Representative photomicrographs of extracellular matrix proteins on murine bone marrow-derived MSC-chitosan scaffold constructs (without HA) incubated in culture medium containing various concentrations of HA for 48 hours or 3 weeks in various conditions and stained for proteoglycans. Scaffolds appear dark, and proteoglycans appear as teal-colored strands. A—Culture medium contained no HA. B—Culture medium contained HA at 0.05 mg/mL. C—Culture medium contained HA at 1.0 mg/mL. D—Culture medium contained HA at 2.0 mg/mL. E—Constructs incubated for 21 days in growth medium without HA. There are no extracellular fibers present. F—Constructs incubated for 21 days in chondrogenic medium containing HA (1.0 mg/mL). Notice the extracellular fibers staining for proteoglycans. Saffranin-O and fast green stain; bar = 50 μm in panels A through D and 10 μm in panels E and F.
Citation: American Journal of Veterinary Research 72, 1; 10.2460/ajvr.72.1.42
Representative photomicrographs of extracellular matrix proteins on murine bone marrow-derived MSC-chitosan scaffold constructs containing HA, immunologically stained to detect aggrecan. Scaffolds stain blue or gray, and aggrecan stains brown. A—Culture medium contained no HA. B—Culture medium contained HA at 0.05 mg/mL. C—Culture medium contained HA at 1.0 mg/mL. D—Culture medium contained HA at 2.0 mg/mL. E—Scaffold construct incubated 21 days in growth medium without HA, in which anti-aggrecan antibody was included during staining. Notice only cells stain lightly for aggrecan with no pericellular fibers. F—Negative control construct (no anti-aggrecan antibody added). G—Scaffold construct incubated 21 days in chondrogenic medium containing HA (2.0 mg/mL), in which anti-aggrecan antibody was included during staining. Notice the pericellular fibers stained positively for aggrecan. H—Negative control construct (no anti-aggrecan antibody added). Bar = 50 μm in panels A through D and 10 μm in panels E through H.
Citation: American Journal of Veterinary Research 72, 1; 10.2460/ajvr.72.1.42
Representative photomicrographs of extracellular matrix proteins on murine bone marrow-derived MSC-chitosan scaffold constructs containing HA, immunologically stained to detect aggrecan. Scaffolds stain blue or gray, and aggrecan stains brown. A—Culture medium contained no HA. B—Culture medium contained HA at 0.05 mg/mL. C—Culture medium contained HA at 1.0 mg/mL. D—Culture medium contained HA at 2.0 mg/mL. E—Scaffold construct incubated 21 days in growth medium without HA, in which anti-aggrecan antibody was included during staining. Notice only cells stain lightly for aggrecan with no pericellular fibers. F—Negative control construct (no anti-aggrecan antibody added). G—Scaffold construct incubated 21 days in chondrogenic medium containing HA (2.0 mg/mL), in which anti-aggrecan antibody was included during staining. Notice the pericellular fibers stained positively for aggrecan. H—Negative control construct (no anti-aggrecan antibody added). Bar = 50 μm in panels A through D and 10 μm in panels E through H.
Citation: American Journal of Veterinary Research 72, 1; 10.2460/ajvr.72.1.42
Representative photomicrographs of extracellular matrix proteins on murine bone marrow-derived MSC-chitosan scaffold constructs containing HA, immunologically stained to detect aggrecan. Scaffolds stain blue or gray, and aggrecan stains brown. A—Culture medium contained no HA. B—Culture medium contained HA at 0.05 mg/mL. C—Culture medium contained HA at 1.0 mg/mL. D—Culture medium contained HA at 2.0 mg/mL. E—Scaffold construct incubated 21 days in growth medium without HA, in which anti-aggrecan antibody was included during staining. Notice only cells stain lightly for aggrecan with no pericellular fibers. F—Negative control construct (no anti-aggrecan antibody added). G—Scaffold construct incubated 21 days in chondrogenic medium containing HA (2.0 mg/mL), in which anti-aggrecan antibody was included during staining. Notice the pericellular fibers stained positively for aggrecan. H—Negative control construct (no anti-aggrecan antibody added). Bar = 50 μm in panels A through D and 10 μm in panels E through H.
Citation: American Journal of Veterinary Research 72, 1; 10.2460/ajvr.72.1.42
Mean ± SE percentage of chitosan scaffolds staining positively for proteoglycan and aggrecan in murine bone marrow-derived MSC-scaffold constructs 21 days after incubation with various concentrations of HA in culture medium. The percentage coverage significantly increased with increasing HA concentration for proteoglycans (P = 0.011) and aggrecan (P < 0.001).
Citation: American Journal of Veterinary Research 72, 1; 10.2460/ajvr.72.1.42
Mean ± SE percentage of chitosan scaffolds staining positively for proteoglycan and aggrecan in murine bone marrow-derived MSC-scaffold constructs 21 days after incubation with various concentrations of HA in culture medium. The percentage coverage significantly increased with increasing HA concentration for proteoglycans (P = 0.011) and aggrecan (P < 0.001).
Citation: American Journal of Veterinary Research 72, 1; 10.2460/ajvr.72.1.42
Mean ± SE percentage of chitosan scaffolds staining positively for proteoglycan and aggrecan in murine bone marrow-derived MSC-scaffold constructs 21 days after incubation with various concentrations of HA in culture medium. The percentage coverage significantly increased with increasing HA concentration for proteoglycans (P = 0.011) and aggrecan (P < 0.001).
Citation: American Journal of Veterinary Research 72, 1; 10.2460/ajvr.72.1.42
Discussion
The main finding of the study reported here was the dose-dependent beneficial effect of medium supplementation with high-MW HA on chondrogenesis by bone marrow-derived MSCs cultured on chitosan scaffolds. The MSCs differentiated into chondrocytes, confirming previous reports38 of chondrogenesis within chitosan sponges, and produced significantly greater amounts of type II collagen, glycosaminoglycans, and aggrecan in the presence of higher medium HA concentrations. Incorporation of HA into hybrid chitosan scaffolds did not appear to affect any of the characteristics tested in our study.
Hyaluronic acid is a usual constituent of synovial fluid, with concentrations ranging from 2 to 3 mg/mL in healthy human knees and decreasing to 0.17 to 1.32 mg/mL in diseased joints.40,41 The situation is similar in dogs, in which synovial fluid of healthy stifle joints reportedly contains a mean HA concentration of 15.3 mg/mL, compared with 1.16 mg/mL in osteoarthritic joints.42 This correlation between joint status and HA concentration supports results of in vitro studies43–46 suggesting the beneficial effects of HA on articular cartilage homeostasis and prompted the study reported here. A high-MW HA was selected for our study because one of the proposed benefits of intra-articular HA treatment is an increase in the viscoelasticity of synovial fluid in the joint. This effect should correlate with the MW of the compound, explaining the clinical superiority of high-MW HA treatment in horses and humans relative to low-MW treatment.26,27
The concentrations of HA evaluated in our study were chosen on the basis of previous experimental and clinical studies.26,27,30,38,43 Among these, incubation of chondrocytes in gelatin containing high-MW HA at a concentration of 0.5 mg/mL induced a 22-fold increase in proteoglycan synthesis.43 The higher dose of 2 mg of HA/mL was selected to approximate the HA concentration achieved in the joints after clinical intra-articular injection of HA in horses.39 In our study, supplementing a chondrogenic culture medium with high-MW HA improved matrix production by bone marrow-derived MSCs in a dose-dependent manner, as revealed through biochemical analysis of constructs (GAG, collagen II, proteoglycan, and aggrecan). Results of histologic examination, although semiquantitative, supported the conclusions obtained from the assays used for GAG and collagen II. This effect was not influenced by the nature of the scaffold (chitosan or hybrid sponges) and did not appear related to an improvement in cell attachment or increase in cell proliferation because construct DNA content did not differ among constructs cultured with or without HA supplementation at 2 or 21 days. Instead, the increase in matrix content we found likely reflected an improvement in chondrogenesis by individual cells. When control scaffold (chitosan and hybrid sponges without MSCs) was evaluated, no GAG or collagen II was detected. These results confirmed previous in vitro evidence that HA stimulates matrix production by chondrocytes.44–47
The magnitude of the positive effect of HA on chondrogenesis may vary with the type of cells evaluated because addition of 2 mg of HA/mL to the culture medium resulted in a 2-fold increase in the GAG:DNA ratio of the MSCs cultured in our study. The influence of HA supplementation together with the addition of autologous synovial fluid can also induce chondrogenesis of equine MSCs in vitro with pronounced proteoglycan expression.48 This effect has been attributed to the interaction between HA and its receptor, CD44, which is expressed by MSCs.49 However, investigators in that study tested a low dose of HA (0.1 mg/mL) on pellets of MSCs, and results were solely obtained from histologic and immunochemical evaluations. Results of the present study provided quantitative data to confirm that HA supplementation enhances chondrogenesis of stem cells when used as an adjunct to a chondrogenic growth factor such as TGF-β1. Although not tested in our study, HA supplementation alone is unlikely to replace the use of chondrogenic growth factors. Indeed, HA alone was recently found to stimulate the expression of chondrogenic markers, collagen type II, and aggrecan by bone marrow-derived stem cells.50 However, the concurrent presence of TGF-β3 was required to increase aggrecan synthesis.50 Indeed, HA appears to play a synergetic role with TGF-β, modulating the chondrogenic phenotype of MSCs via a reduction in collagen type I and X production.48,50
If HA supplementation improved chondrogenesis in both types of sponges tested in the present study, then the presence of HA within the scaffold had little effect on MSCs. Our research group previously reported that chondrocytes maintained their phenotype and produced more GAG and collagen II when cultured on chitosan sponges rather than a polyglycolic acid fiber mesh.18 Nevertheless, the addition of HA in the hybrid sponges we used did not improve chondrogenesis (as judged via biochemical test results) or MSC attachment (based on DNA quantification 48 hours after cell seeding) relative to that of the pure chitosan scaffolds of identical structure. The only potential benefit of adding HA to the scaffold was a nonsignificant increase in cell viability and proliferation detected via viability assays and DNA quantification 21 days after cell seeding. The discrepancy between the effects of HA as a component of the culture medium or the scaffold may have resulted from a lack of exposure of cells to the HA incorporated in the matrix. This lack of exposure may have resulted from the low dose of HA (0.01%) incorporated in hybrid scaffolds. Our ability to increase the concentration of HA in chitosan scaffolds was limited by the rapid dissolution of scaffolds. Hybrid sponges containing 0.1% HA spontaneously dissolve within 48 hours after contact with culture medium, deeming them impractical for tissue engineering purposes. The sponges evaluated in our study remained intact after 21 days of contact with culture medium devoid of cells.
The second mechanism by which cells may not have gained adequate access to HA in hybrid scaffolds may relate to differences in molecular configuration. Studies44–46 have been conducted to test the dose-dependent effects of HA bound to scaffolds or gels through use of rabbit or bovine chondrocytes in alginate or collagen matrices. Similar to our study, these studies44–46 found that higher concentrations of bound HA increased proliferation but decreased ECM protein secretion and accumulation by the inhibition of conversion of the low-affinity form of proteoglycans to a high-affinity form through addition of HA. Hyaluronan is one of the key components of the articular cartilage matrix and synovial fluid51 and is necessary for cells to construct ECM in the early stages of their expansion and differentiation. In addition, hyaluronan forms a polyion complex with collagen, allowing the fixation and assembly of collagen and aggrecan molecules after their secretion by the cells.52,53 It is possible that the HA in the surrounding medium is more available for use in constructing these complexes than that bound to the scaffolds.
The third potential cause for a decreased exposure of cells to HA contained in the matrix relates to the cell distribution in these constructs. Indeed, cells remained grouped around the periphery of the scaffold and commonly formed clusters, preventing contact between cells and HA contained in the matrix. Seeding in an orbital shaker was selected in this study as a cost-effective dynamic seeding technique allowing individual tracking of scaffolds and flexibility in selecting scaffold numbers (sample size). Although not established for MSCs, techniques relying on a vacuum chamber and perfused bioreactor or a spinner flask may have improved the uniformity of cell distribution throughout the scaffolds and improved the contact between cells and matrix.
In the study reported here, the addition of HA to a chondrogenic medium increased ECM production by differentiated MSCs in chitosan and hybrid chitosan-HA sponges in a dose-dependent manner. The addition of HA in the surrounding fluid during chondrogenesis and cartilage production should improve engineered cartilage tissues for use in replacement treatments. The optimal additive dose of HA remains undetermined. Additional research is warranted to determine the optimal dose for supplementation of HA, verify the effects in primary cells of relevant animal species, and elucidate the mechanism of action of HA on MSCs.
ABBREVIATIONS
| DMEM | Dulbecco modified Eagle medium |
| ECM | Extracellular matrix |
| GAG | Glycosaminoglycan |
| HA | Hyaluronic acid |
| MSC | Mesenchymal stem cell |
| MW | Molecular weight |
| SEM | Scanning electron microscopy |
| TGF | Transforming growth factor |
D1 ORL UVA, American Type Culture Collection, Manassas, Va.
American Type Culture Collection, Manassas, Va.
Sigma Chemical Co, St Louis, Mo.
1% ITS Premix, Collaborative Biomedical Products, Bedford, Mass.
TGFβ-1, 10 ng/mL, recombinant human, R&D Systems, Minneapolis, Minn.
Vanson Inc, Redmond, Wash.
Hylartin V, Pfizer Animal Health, New York, NY.
LIVE/DEAD Viability/Cytotoxicity Kit, Invitrogen, Carlsbad, Calif.
Olympus BX50 Confocal Microscope, Olympus, Center Valley, Pa.
Fluoview, version FV300, Olympus, Center Valley, Pa.
Hoechst 33258, Sigma-Aldrich, St Louis, Mo.
Fluostar Optima Spectrophotometer, BMG Lab Technologies, Durham, NC.
DMMB GAG Assay, Sigma-Aldrich, St Louis, Mo.
Type II collagen detection kit, Chondrex Inc, Redmond, Wash.
Hitachi S4700, Hitachi, Schaumburg, Ill.
Veterinary Biosciences Histology Laboratory, University of Illinois, Urbana, Ill.
Image J program, National Institutes of Health, Bethesda, Md.
PROC MIXED, SAS, version 9.2, SAS Institute Inc, Cary, NC.
Stata, version 8.0, Stata Corp, College Station, Tex.
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