Combined effects of insulin-like growth factor-1 and transforming growth factor-beta1 on periosteal mesenchymal cells during chondrogenesis in vitro

OBJECTIVE: Periosteum contains undifferentiated mesenchymal stem cells that have both chondrogenic and osteogenic potential, and has been used to repair articular cartilage defects. During this process, the role of growth factors that stimulate the periosteal mesenchymal cells toward chondrogenesis to regenerate articular cartilage and maintain its phenotype is not yet fully understood. In this study, we examined the effects of insulin-like growth factor-1 (IGF-1) and transforming growth factor-beta1 (TGF-beta1), alone and in combination, on periosteal chondrogenesis using an in vitro organ culture model. METHODS: Periosteal explants from the medial proximal tibia of 2-month-old rabbits were cultured in agarose under serum free conditions for up to 6 weeks. After culture the explants were weighed, assayed for cartilage production via Safranin O staining and histomorphometry, assessed for proliferation via proliferative cell nuclear antigen (PCNA) immunostaining, and assessed for type II collagen mRNA expression via in situ hybridization. RESULTS: IGF-1 significantly increased chondrogenesis in a dose-dependent manner when administered continuously throughout the culture period. Continuous IGF-1, in combination with TGF-beta1 for the first 2 days, further enhanced overall total cartilage growth. Immunohistochemistry for PCNA revealed that combining IGF-1 with TGF-beta1 gave the strongest proliferative stimulus early during chondrogenesis. In situ hybridization for type II collagen showed that continuous IGF-1 maintained type II collagen mRNA expression throughout the cambium layer from 2 to 6 weeks. CONCLUSION: The results of this study demonstrate that IGF-1 and TGF-beta1 can act in combination to regulate proliferation and differentiation of periosteal mesenchymal cells during chondrogenesis.     

Viability of periosteal tissue obtained postmortem

Periosteal autografts have the potential to regenerate articular cartilage defects, but this potential is limited by the patient's age. Allograft transplantation from a young donor to an older recipient might bypass this limitation. The effect of the time delay, between death and harvesting of a periosteal graft, on the chondrogenic potential of periosteum is important not only for transplantation but also for studies dealing with tissues retrieved postmortem (i.e., including the periosteal explant model). The purpose of this study was to investigate the chondrogenic potential of periosteum obtained postmortem and a possible beneficial effect of hypothermia. Thirty NZ white rabbits (2 months old) were sacrificed and stored at room temperature or 4 degrees C for 0, 4, 6, 8, 12, 16, 18, or 24 h. Periosteal explants were then obtained and a standard cartilage yield assay performed by culturing them for 6 weeks using the periosteal organ culture model as previous published. TGF-beta1 (10 ng/ml) was added for the first 14 days of culture. Histochemical analysis and quantitative collagen typing were performed. In the explants from the animals kept for 4 h at room temperature growth and chondrogenesis were dramatically reduced. Little or no chondrogenesis was seen in explants from rabbits maintained at room temperature after 4-8 h (or more) postmortem. Cooling the rabbits to 4 degrees C partially prevented this loss of viability and continued to do so for 24 h. Even storage at 4 degrees C did not eliminate the decrease in chondrogenic potential, though it did permit partial preservation of chondrogenic potential. If periosteum is to be used for allograft transplantation, or if it is used for experimental study, its viability must be assured. This is best accomplished by harvesting it immediately postmortem. Preservation techniques, cryopreservation, or hypothermia might be useful in preserving periosteal chondrogenic potential.     

Insulin is essential for in vitro chondrogenesis of mesenchymal progenitor cells and influences chondrogenesis in a dose-dependent manner

Purpose

Insulin is a commonly used additive in chondrogenic media for differentiating mesenchymal stem cells (MSCs). The indispensability of other bioactive factors like TGF-β or dexamethasone in these medium formulations has been shown, but the role of insulin is unclear. The purpose of this study was to investigate whether insulin is essential for MSC chondrogenesis and if there is a dose-dependent effect of insulin on MSC chondrogenesis.

Methods

We cultivated human MSCs in pellet culture in serum-free chondrogenic medium with insulin concentrations between 0 and 50 μg/ml and assessed the grade of chondrogenic differentiation by histological evaluation and determination of glycosaminoglycan (GAG), total collagen and DNA content. We further tested whether insulin can be delivered in an amount sufficient for MSC chondrogenesis via a drug delivery system in insulin-free medium.

Results

Chondrogenesis was not induced by standard chondrogenic medium without insulin and the expression of cartilage differentiation markers was dose-dependent at insulin concentrations between 0 and 10 μg/ml. An insulin concentration of 50 μg/ml had no additional effect compared with 10 μg/ml. Insulin was delivered by a release system into the cell culture under insulin-free conditions in an amount sufficient to induce chondrogenesis.

Conclusions

Insulin is essential for MSC chondrogenesis in this system and chondrogenic differentiation is influenced by insulin in a dose-dependent manner. Insulin can be provided in a sufficient amount by a drug delivery system. Therefore, insulin is a suitable and inexpensive indicator substance for testing drug release systems in vitro.     

Temporal expression patterns of BMP receptors and collagen II (B) during periosteal chondrogenesis.

Articular cartilage has a limited ability to repair itself. Periosteal grafts have chondrogenic potential and are used clinically to repair defects in articular cartilage. An organ culture model system for in vitro rabbit periosteal chondrogenesis has been established to study the molecular events of periosteal chondrogenesis in vitro. In this model, bone morphogenetic protein-2 (BMP2) mRNA expression was found to be upregulated in the first 12 h. BMPs usually transduce their signals through a receptor complex that includes type II and either type IA or type IB BMP receptors. Receptors IA and IB play distinct roles during limb development. We have examined the temporal expression patterns for the mRNAs of these receptors using our experimental model. The mRNA expression patterns of these three BMP receptors differed from one another in periosteal explants during chondrogenesis. When these explants were cultured under chondrogenic conditions (agarose suspension with TGF-beta1 added to the media for the first 2 days), the expression of BMPRII mRNA and that of BMPRIA mRNA varied only slightly and persisted over a long time. In contrast, the expression of BMPRIB mRNAwas upregulated within 12 h, peaked at day 5, and fell to a level that was barely detected beyond day 21. Moreover, the expression of BMPRIB mRNA preceded that of collagen type IIB mRNAs, a marker for matrix-depositing chondrocytes. These data support a role for coordinate expression of BMP2 and its receptors early during periosteal chondrogenesis.     

Synergistic effect of growth factors on cell outgrowth from explants of rabbit anterior cruciate and medial collateral ligaments

Cellular migration and proliferation are integral aspects of wound healing. An in vitro assay for cellular migration and proliferation using explants of rabbit anterior cruciate and medial collateral ligaments was developed previously. This study presents the effects of serum-free culture medium supplemented with basic fibroblast growth factor, bovine insulin, transforming growth factor-β1, and platelet-derived growth factor-B, added either individually or in combination, on cell outgrowth in explants of rabbit anterior cruciate and medial collateral ligaments. Outgrowth was assessed at 3 and 6 days by counting the number of cells surrounding the tissue explants. For explants of both ligaments, cell outgrowth was dependent on the presence of 10% fetal bovine serum or the combination of growth factors. Little outgrowth occurred in explants of either ligament in the presence of basic fibroblast growth factor, transforming growth factor-β1, or bovine insulin. Platelet-derived growth factor-B at concentrations of 20 and 100 ng/ml seemed to increase cell outgrowth from medial collateral ligament explants at 6 days. The outgrowth from the explants of both ligaments was much greater in the presence of all four growth factors than the sum of the outgrowth with the individual factors. This synergistic effect was as much as 10-fold and 20-fold for the anterior cruciate ligament explants at days 3 and 6, respectively, but no more than 3-fold for the medial collateral ligament explants at these times. Medial collateral ligament explants exhibited greater cell outgrowth than anterior cruciate ligament explants in 10% serum and in the presence of the four growth factors.     

The enhancement of periosteal chondrogenesis in organ culture by dynamic fluid pressure.

Cartilage repair by autologous periosteal arthroplasty is enhanced by continuous passive motion (CPM) of the joint after transplantation of the periosteal graft. However, the mechanisms by which CPM stimulate chondrogenesis are unknown. Based on the observation that an oscillating intra-synovial pressure fluctuation has been reported to occur during CPM (0.6-10 kPa), it was hypothesized that the oscillating pressure experienced by the periosteal graft as a result of CPM has a beneficial effect on the chondrogenic response of the graft. We have developed an in vitro model with which dynamic fluid pressures (DFP) that mimic those during CPM can be applied to periosteal explants while they are cultured in agarose gel suspension. In this study periosteal explants were treated with or without DFP during suspension culture in agarose, which is conducive to chondrogenesis. Different DFP application times (30 min, 4 h, 24 h/day) and pressure magnitudes (13, 103 kPa or stepwise 13 to 54 to 103 kPa) were compared for their effects on periosteal chondrogenesis. Low levels of DFP (13 kPa at 0.3 Hz) significantly enhanced chondrogenesis over controls (34 +/- 7% vs 14 +/- 5%; P < 0.05), while higher pressures (103 kPa at 0.3 Hz) completely inhibited chondrogenesis, as determined from the percentage of tissue that was determined to be cartilage by histomorphometry. Application of low levels of DFP to periosteal explants also resulted in significantly increased concentrations of Collagen Type II protein (43 +/- 8% vs 10 +/- 5%; P < 0.05). New proteoglycan synthesis, as measured by 35S-sulphate uptake was increased by 30% in periosteal explants stimulated with DFP (350 +/- 50 DPM vs 250 +/- 75 DPM of 35S-sulphate uptake/microg total protein), when compared to controls though this difference was not statistically significant. The DFP effect at low levels was dose-dependant for time of application as well, with 4 h/day stimulation causing significantly higher chondrogenesis than just 30 min/day (34 +/- 7 vs 12 +/- 4% cartilage; P < 0.05) and not significantly less than that obtained with 24 h/day of DFP (48 +/- 9% cartilage, P > 0.05). These observations may partially explain the beneficial effect on cartilage repair by CPM. They also validate an in vitro model permitting studies aimed at elucidating the mechanisms of action of mechanical factors regulating chondrogenesis. The fact that these tissues were successfully cultured in a mechanical environment for six weeks makes it possible to study the actions of mechanical factors on the entire chondrogenic pathway, from induction to maturation. Finally, these data support the theoretical predictions regarding the role of hydrostatic compression in fracture healing.     

Influence of 17beta-estradiol and insulin on type II collagen and protein synthesis of articular chondrocytes

Clinical observations have suggested a relationship between osteoarthritis and a changed estrogen metabolism in menopausal women. Type II collagen is one main structural protein of articular cartilage matrix and its synthesis is increased by insulin in growth plate cartilage. Therefore, it was investigated if [(3)H]-proline incorporation and type II collagen synthesis (immunocytochemistry, ELISA) in female bovine articular chondrocytes are affected by 17beta-estradiol and/or insulin. Articular chondrocytes were cultured in monolayers at 5% O(2) in medium containing serum for 5-9 days, followed by application of 10(-13) to 10(-9) M estradiol or 5 microg/ml insulin during a serum-free culture phase of 2-3 days. Immunostaining for type II collagen was strong in the serum-free culture phase whereas it was negative for type I collagen, indicating that cells did not dedifferentiate to fibroblast-like cells during culture in serum-free medium. Whereas insulin raised the proline incorporation and the type II collagen synthesis significantly, physiological doses of estradiol did not show significant effects. The stimulating effect of insulin on the [(3)H]-proline incorporation or the type II collagen synthesis was significantly suppressed after preincubation of cells with 10(-11) to 10(-9) M estradiol resembling an unfavorable effect for articular cartilage. The suppression was reversed if cells were incubated with 10(-11) to 10(-7) M tamoxifen or ICI 182,780 combined with 10(-11) or 10(-9) M estradiol followed by incubation with 5 microg/ml insulin, indicating an estrogen receptor-mediated process. Because the articular cartilage of diabetic patients is biomechanically less stable, further experiments are needed to clarify the role of estradiol and insulin in the metabolism of articular chondrocytes.     

Culturing periosteum in vitro: the influence of different sizes of explants

Periosteal transplantation is being used clinically to repair articular defects. Isolated cells and very small periosteal explants can be grown in tissue culture, but it will be necessary to test larger sizes for tissue engineering to be applied to clinical transplantation of periosteum. This study was conducted to assess the chondrogenic potential of different sizes of periosteal explants in agarose culture. Ninety-six rabbit tibial periosteal explants in three different sizes (small 1.5 × 2, medium 3 × 2, and large 4 × 6 mm, 32 pieces per size) were cultured in agarose suspension for 6 wk and given TGF-β1 (10 ng/mL) for the first 2 wk. Tissue growth, as indicated by normalized final wet weights of the explants after 6 wk in culture, was inversely proportional to explant size. Cartilage formation was observed in all explants. Histomorphometry revealed that cartilage formation was significantly better for the smaller explants (80% cartilage), but similar in the medium and larger explants (60% cartilage). Similar proportions of type II collagen were present in the different-sized explants. This study demonstrates that various sizes of periosteal explants can be grown in culture. Abundant cartilage was produced even by the large explants.     

Relationship of donor site to chondrogenic potential of periosteum in vitro

Periosteum has been shown in vitro and in vivo to have a chondrogenic potential that permits it to be used for cartilage regeneration. A useful donor site should have good chondrogenic potential, availability of a large quantity of periosteum, and relative ease of access, and it should be associated with a low rate of morbidity. We hypothesized that the chondrogenic potential of periosteum varies from one bone to another and among different regions of the periosteum from a single bone. A total of 370 periosteal and 37 fascia lata (control) explants were taken from the skull, the ilium, the scapula, the upper, middle, and lower medial proximal tibia, the posterior proximal tibia, and the distal tibia of 2-month-old New Zealand rabbits. The explants were cultured for 6 weeks in agarose/Dulbecco's modified Eagle medium to which 10 ng/ml of transforming growth factor-β1 was added during the first 2 weeks. Skeletal muscle and fascia lata were used as controls. In addition, the thickness, cell density, and total cell count of the cambium layer were measured in 24 explants from the donor sites on the ilium and the upper, middle, and lower proximal tibia. At 6 weeks, histomorphometry and quantitative collagen typing were performed. The periosteal donor sites could be grouped into three categories according to chondrogenic potential: ilium (best), scapula and tibia, and skull (no chondrogenesis). The scapular periosteum was slightly better than that from the tibia. Within the tibia, the upper and middle zones of the proximal region were similar and were slightly better than the lower proximal tibia or the distal tibia. The cellularity of the cambium layer correlated positively with the amount of cartilage as a percentage of the total area. The results of this study indicate that iliac periosteum exhibited the best overall chondrogenic potential in vitro but that periosteum from the traditionally used medial proximal tibia also was excellent. Periosteum from the skull was not chondrogenic. The chondrogenic potential of periosteum varies from bone to bone and within the periosteum from one bone. This variation in chondrogenic potential among donor sites may be due to a difference in the total cell count of the cambium layer.     

Long-term explant culture of rabbit flexor tendon: effects of recombinant human insulin-like growth factor-I and serum on matrix metabolism

The effects of human recombinant insulin-like growth factor-I (rhIGF-I, 50 ng/ml) on matrix metabolism in the deep flexor tendon from the tendon sheath region of the rabbit were studied in explants cultured for 3 weeks. Tendon segments cultured in medium supplemented with fetal calf serum (FCS) exhibited proliferation of the superficial cell layers. Synthesis of proteoglycan and non-collagen protein (NCP) increased threefold during the first week and remained elevated during the next 2 weeks of culture in medium supplemented with rhIGF-I or FCS, but not in medium without supplements (bovine serum albumin, BSA). The estimated halflife (t1/2) for elimination of newly labeled proteoglycans from the tendon explants ranged from 5.1 to 8.5 days and from 4.9 to 6.8 days for NCP in supplemented medium. Presence of rhIGF-I or FCS did not affect degradation of matrix as compared with BSA. The total hexosamine content per tendon segment was stable during the culture period, but the non-collagen protein content decreased by 25%. Collagen synthesis decreased to 10% of the initial level after 3 weeks in supplemented medium, but to 3% in unsupplemented medium. There was no measurable turnover of collagen in explants cultured in either medium, and the collagen content remained unchanged. Our results suggest that rhIGF-I, as well as FCS, stimulates matrix synthesis but does not influence matrix turnover in rabbit flexor tendon explants in long-term culture as compared with medium without supplements. We conclude that rhIGF-I may be used as a defined growth-promoting factor in serum-free media and may be of importance in tendon healing.     

Induction of CD-RAP mRNA during periosteal chondrogenesis.

Induction of chondrogenesis and maintenance of the chondrocyte phenotype are critical events for autologous periosteal transplantation, which is a viable approach for cartilage repair. Cartilage-derived retinoic acid-sensitive protein (CD-RAP) is a recently discovered protein that is mainly produced in cartilage. During development, CD-RAP expression starts at the beginning of chondrogenesis and continues throughout cartilage maturation. In order to investigate the involvement of CD-RAP during periosteal chondrogenesis we have determined the nucleotide sequence of the rabbit CD-RAP mRNA and utilized this information to evaluate the temporal and spatial expression pattern of CD-RAP at the mRNA level during chondrogenesis. When the periosteal explants were cultured under chondrogenic conditions, the expression of CD-RAP was induced, as shown by a 40-fold increase in CD-RAP mRNA between days 7 and 10. The temporal expression pattern of CD-RAP closely mimicked that of collagen type IIB mRNA. Also, the CD-RAP mRNA was localized to the matrix forming chondrocytes in the cambium layer of the periosteum by in situ hybridization as indicated by colocalization with collagen type II mRNA and positive safranin O staining. These data suggest a regulatory role of CD-RAP in periosteal chondrogenesis, which is potentially important for both cartilage repair and fracture healing via callus formation.     

Brief exposure to high-dose transforming growth factor-beta1 enhances periosteal chondrogenesis in vitro: a preliminary report

BACKGROUND: Articular cartilage has limited potential for repair. There have been various attempts aimed at improving the repair process in articular cartilage. Transforming growth factor-beta1 (TGF-beta1) has a stimulatory effect on chondrogenesis in periosteal explants. The purpose of the present study was to determine the effect of brief exposures (i.e., thirty and sixty minutes) of high concentrations of TGF-beta1 on periosteal chondrogenesis. METHODS: Five hundred and seventy-three periosteal explants were harvested from forty-six two-month-old male New Zealand White rabbits. Explants were exposed to 50 or 100 ng/mL of TGF-beta1 for thirty or sixty minutes. The amount of cartilage formed was then determined with use of a standardized six-week agarose culture assay. RESULTS: There was a significant increase in the amount of cartilage formation (p < 0.01), Type-II collagen content (p < 0.05), and sulfate incorporation (p < 0.0001) in explants treated with TGF-beta1. Maximal stimulation occurred following exposure to 100 ng/mL of TGF-beta1 for thirty minutes. There was also an increase in chondrocyte proliferation as measured by [ (3) H-] thymidine incorporation on day 5 of culture (p < 0.049). Conclusions: The findings of this study indicate that exposure to TGF-beta1 has a stimulatory effect on periosteal chondrogenesis. This stimulatory effect is observed even with a very brief exposure time of thirty minutes. Clinical Relevance: A possible clinical application of these findings is exposure of periosteal grafts that are currently utilized clinically to resurface articular defects to TGF-beta1 during the short time between graft procurement and implantation into the joint. This may obviate the need for intra-articular administration of TGF-beta1 and may enhance the ultimate graft incorporation and quality of cartilage repair.     

Periosteum responds to dynamic fluid pressure by proliferating in vitro.

Periosteum provides a source of undifferentiated chondrocyte precursor cells for fracture healing that can also be used for cartilage repair. The quantity of cartilage that can be produced, which is a determining factor in fracture healing and cartilage repair, is related to the number of available stem cells in the cambium layer. Cartilage formation during both of these processes is enhanced by motion of the fracture or joint in which periosteum has been transplanted. The effect of dynamic fluid pressure on cell proliferation in periosteal tissue cultures was determined in 452 explants from 60 immature (2-month-old) New Zealand White rabbits. The explants were cultured in agarose suspension for 1-14 days. One group was subjected to cyclic hydrostatic pressure, which is referred to as dynamic fluid pressure, at 13 kPa and a frequency of 0.3 Hz. Control explants were cultured in similar chambers without application of pressure. DNA synthesis ([3H]thymidine uptake) and total DNA were measured. The temporal pattern and distribution of cell proliferation in periosteum were evaluated with autoradiography and immunostaining with proliferating cell nuclear antigen. Dynamic fluid pressure increased proliferation of periosteal cells significantly, as indicated by a significant increase in [3H]thymidine uptake at all time points and a higher amount of total DNA compared with control values. On day 3, when DNA synthesis reached a peak in periosteal explants, [3H]thymidine uptake was 97,000+/-5,700 dpm/microg DNA in the group exposed to dynamic fluid pressure and 46,000+/-6,000 dpm/microg in the controls (p < 0.001). Aphidicolin, which blocks DNA polymerase alpha, inhibited [3H]thymidine uptake in a dose-dependent manner in the group subjected to dynamic fluid pressure as well as in the positive control (treated with 10 ng/ml of transforming growth factor-beta1) and negative control (no added growth factors) groups, confirming that [3H]thymidine measurements represent proliferation and dynamic fluid pressure stimulates DNA synthesis. Total DNA was also significantly higher in the group exposed to dynamic fluid pressure (5,700+/-720 ng/mg wet weight) than in the controls (3,700+/-630) on day 3 (p < 0.01). Autoradiographs with [3H]thymidine revealed that one or two cell cycles of proliferation took place in the fibrous layer prior to proliferation in the cambium layer (where chondrocyte precursors reside). Proliferating cell nuclear antigen immunophotomicrographs confirmed the increased proliferative activity due to dynamic fluid pressure. These findings suggest either a paracrine signaling mechanism between the cells in these two layers of the periosteum or recruitment/migration of proliferating cells from the fibrous to the cambium layer. On the basis of the data presented in this study, we postulate that cells in the fibrous layer respond initially to mechanical stimulation by releasing growth factors that induce undifferentiated cells in the cambium layer to divide and differentiate into chondrocytes. These data indicate that cell proliferation in the early stages of chondrogenesis is stimulated by mechanical factors. These findings are important because they provide a possible explanation for the increase in cartilage repair tissue seen in joints subjected to continuous passive motion. The model of in vitro periosteal chondrogenesis under dynamic fluid pressure is valuable for studying the mechanisms by which mechanical factors might be involved in the formation of cartilage in the early fracture callus and during cartilage repair.     

Chondrogenic potential of human synovial mesenchymal stem cells in alginate

OBJECTIVE: In a recent study, we demonstrated that mesenchymal stem cells (MSCs) derived from the synovial membranes of bovine shoulder joints could differentiate into chondrocytes when cultured in alginate. The purpose of the present study was to establish the conditions under which synovial MSCs derived from aging human donors can be induced to undergo chondrogenic differentiation using the same alginate system. METHODS: MSCs were obtained by digesting the knee-joint synovial membranes of osteoarthritic human donors (aged 59-76 years), and expanded in monolayer cultures. The cells were then seeded at a numerical density of 4x10(6)/ml within discs of 2% alginate, which were cultured in serum-containing or serum-free medium (the latter being supplemented with 1% insulin, transferrin, selenium (ITS). The chondrogenic differentiation capacity of the cells was tested by exposing them to the morphogens transforming growth factor-beta1 (TGF-beta1), TGF-beta2, TGF-beta3, insulin-like growth factor-1 (IGF-1), bone morphogenetic protein-2 (BMP-2) and BMP-7, as well as to the synthetic glucocorticoid dexamethasone. The relative mRNA levels of collagen types I and II, of aggrecan and of Sox9 were determined quantitatively by the real-time polymerase chain reaction (PCR). The extracellular deposition of proteoglycans was evaluated histologically after staining with Toluidine Blue, and that of type-II collagen by immunohistochemistry. RESULTS: BMP-2 induced the chondrogenic differentiation of human synovial MSCs in a dose-dependent manner. The response elicited by BMP-7 was comparable. Both of these agents were more potent than TGF-beta1. A higher level of BMP-2-induced chondrogenic differentiation was achieved in the absence than in the presence of serum. In the presence of dexamethasone, the BMP-2-induced expression of mRNAs for aggrecan and type-II collagen was suppressed; the weaker TGF-beta1-induced expression of these chondrogenic markers was not obviously affected. CONCLUSIONS: We have demonstrated that synovial MSCs derived from the knee joints of aging human donors possess chondrogenic potential. Under serum-free culturing conditions and in the absence of dexamethasone, BMP-2 and BMP-7 were the most potent inducers of this transformation process.     

Mixtures of glucosamine and chondroitin sulfate reverse fibronectin fragment mediated damage to cartilage more effectively than either agent alone

OBJECTIVE: To test the effectiveness of glucosamine (GluNH(2))-HCl, chondroitin sulfate (CS) and mixtures in protecting cartilage exposed to fibronectin fragments (Fn-fs), an exposure known to enhance catabolic cytokines and matrix metalloproteinases (MMPs). METHODS: Pharmacologic formulations of GluNH(2) (FCHG49) and CS (TRH122) (Nutramax Laboratories, Inc.) were added at 1, 10 or 100 microg/ml singly or in mixtures to bovine cartilage cultures in serum or serum-free conditions with or without Fn-f. Proteoglycan (PG) release into media and remaining cartilage PG content were measured by dye binding analysis and effects on PG synthesis by assays of 35-sulfate incorporation. Effects on MMP-3 and -13 expression were measured by Western blotting of conditioned media. RESULTS: In serum-free conditions, the agents singly or as mixtures did not block Fn-f mediated matrix degradation. In serum, single agents were weakly effective at 100 microg/ml, while the mixture of each agent at 0.1 microg/ml decreased PG loss by about 50% by day 7 and at 1 microg/ml restored nearly 50% of the PG after 7 days in Fn-f pretreated cartilage. However, both agents singly and as mixtures at 0.1-100 microg/ml decreased MMP release. In serum, the single agents at 1-10 microg/ml weakly reversed Fn-f mediated PG synthesis suppression, while the mixtures were 100% effective at 1 microg/ml. CONCLUSIONS: GluNH(2) and CS act synergistically in reversing damage and promoting repair at concentrations found in plasma after oral ingestion of these agents. Reversal of PG synthesis suppression correlates more with these activities than suppression of MMP-3 or -13 expression.     

Effects of basic fibroblast growth factor,transforming growth factor-β1, insulin-like growth factor-1, and insulin on human osteoarthritic articular cartilage explants

This study evaluated the effects of basic fibroblast growth factor, transforming growth factor-β1, insulin-like growth factor-1, and insulin on the incorporation of thymidine and sulfate in human osteoarthritic articular cartilage. Tissue explants were obtained from 11 patients undergoing total knee arthroplasty and were categorized as nonfibrillated or fibrillated cartilage. The explants were cultured for 22 days, with changes of medium and growth factor every 72 hours, and labeled with [³H]thymidine and [³⁵S]sulfate. Growth factors were used in the following concentrations: basic fibroblast growth factor at 1, 10, and 100 ng/ml; transforming growth factor-β1 at 0.5, 5, and 50 ng/ml; insulin-like growth factor-1 at 0.15, 1.5, and 15 ng/ml; and insulin at 0.05, 0.5, and 5 μg/ml. Basic fibroblast growth factor decreased thymidine incorporation to 70% and sulfate incorporation to less than 20% that of the growth factor-free controls. Transforming growth factor-β1 had no significant effect on thymidine incorporation, whereas the concentrations studied inhibited sulfate incorporation to approximately 40% that of the controls. At the concentrations tested, insulin-like growth factor-1 had no significant effect on incorporation of either thymidine or sulfate. In contrast, insulin significantly stimulated the incorporation of both. Compared with growth factor-free controls, insulin maximally increased thymidine incorporation by a factor (± EM) of 2.36 ± 0.47 and 1.69 ± 0.19 in nonfibrillated and fibrillated explants, respectively; sulfate incorporation was maximally increased 1.60 ± 0.24 and 1.92 ± 0.29-fold for nonfibrillated and fibrillated explants, respectively. Of the factors tested, insulin demonstrated the greatest promise for promoting a synthetic response that may contribute to the regeneration of osteoarthritic cartilage.     

The importance of procedure specific training in harvesting periosteum for chondrogenesis

This study was performed to determine the influence of procedure specific and nonspecific training on the chondrogenic potential of explanted periosteum. Seven operators, with varying degrees of orthopaedic surgical experience and procedure specific training in periosteal harvesting, harvested 10 to 16 periosteal explants each from the proximal medial tibiae of 42 New Zealand White rabbits that were 2 months of age. The chondrogenic index assay involved culturing the explants in agarose suspension for 6 weeks, followed by computerized histomorphometric analysis. Chondrogenic indices (the average percent area of cartilage grown in the cultured explants) ranged from 12% to 81% and were influenced strongly by each operator's experience with the technique of periosteal harvesting. Average cartilage yields before practice were in the range of 12% +/- 4% for a technician and 44% +/- 6% for a surgeon, compared with 54% +/- 7% and 79% +/- 2%, respectively, after practice involving more than 300 explants each. Procedure specific experience (with the technique of periosteal harvesting) was more important than the academic qualifications or years of surgical experience in general. These data must be considered when planning or interpreting the results of studies involving periosteal explantation or grafting, or when periosteum serves as a source of mesenchymal stem cells.     

The immunosuppressant FK506 promotes development of the chondrogenic phenotype in human synovial stromal cells via modulation of the Smad signaling pathway

OBJECTIVE: To assess the effect of the immunosuppressant FK506 on chondrogenic differentiation of human synovial stromal cells (hSSCs). METHODS: hSSCs were isolated from synovium of the knee joint and 2x10(5) cells were subjected to pellet culture in chondrogenic culture medium for 3 weeks with or without growth factors [bone morphogenetic protein 2 (BMP2) or transforming growth factor beta(1) (TGFbeta(1))] and +/- addition of FK506 in chondrogenic culture media was evaluated. Chondrogenesis was assessed by the size of the pellet, the production of proteoglycans, and messenger RNA (mRNA) levels for chondrogenic markers. Furthermore, levels and intracellular location of phosphorylated Smad proteins related to BMP signaling and TGFbeta signaling were evaluated following exposure to FK506. RESULTS: FK506 enhanced the differentiation of hSSCs toward a chondrogenic phenotype in a dose-dependent manner associated with increases in glycosaminoglycan synthesis and increased mRNA levels for chondrogenic marker genes. Additionally, FK506 further enhanced chondrogenesis of synovial stromal cells (SSCs) induced by BMP2 and TGFbeta(1), also in a dose-dependent manner. Notably, phosphorylation of Smad1/5/8 and Smad3 was significantly increased by FK506. Also, the ratio of nuclear translocation to cytoplasmic levels of phosphorylated Smad1/5/8 and Smad3 were increased following exposure of SSCs to FK506. Moreover, inhibition of Smad signaling significantly abrogated FK506-induced chondrogenic differentiation of SSCs. CONCLUSION: This study demonstrated that FK506 promotes chondrogenic differentiation of hSSCs likely via impact on Smad signaling pathways. With further optimization, FK506 could potentially be a unique therapeutic tool to promote cartilage repair in clinical situations, as well as enhance development of tissue engineered cartilage in vitro.     

Effect of culture duration on chondrogenic preconditioning of equine bone marrow mesenchymal stem cells in self‐assembling peptide hydrogel

Ex vivo induction of chondrogenesis is a promising approach to improve upon the use of bone marrow mesenchymal stem cells (MSCs) for cartilage tissue engineering. This study evaluated the potential to induce chondrogenesis with days of culture in chondrogenic medium for MSCs encapsulated in self‐assembling peptide hydrogel. To simulate the transition from preconditioning culture to implantation, MSCs were isolated from self‐assembling peptide hydrogel into an individual cell suspension. Commitment to chondrogenesis was evaluated by seeding preconditioned MSCs into agarose and culturing in the absence of the chondrogenic cytokine transforming growth factor beta (TGFβ). Positive controls consisted of undifferentiated MSCs seeded into agarose and cultured in medium containing TGFβ. Three days of preconditioning was sufficient to produce chondrogenic MSCs that accumulated ～75% more cartilaginous extracellular matrix than positive controls by day 17. However, gene expression of type X collagen was ～65‐fold higher than positive controls, which was attributed to the absence of TGFβ. Potential induction of immunogenicity with preconditioning culture was indicated by expression of major histocompatibility complex class II (MHCII), which was nearly absence in undifferentiated MSCs, and ～7% positive for preconditioned cells. These data demonstrate the potential to generate chondrogenic MSCs with days of self‐assembling peptide hydrogel, and the ability to readily recover an individual cell suspension that is suited for injectable therapies. However, continued exposure to TGFβ may be necessary to prevent hypertrophy indicated by type X collagen expression, while immunogenicity may be a concern for allogeneic applications. © 2018 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 37:1368–1375, 2019.