Mechanisms of TGFß3 Action as a Therapeutic Agent for Promoting the Synthesis of Extracellular Matrix Proteins in Hyaline Cartilage.

Hyaline cartilage is a nonvascular connective tissue covering the joint surface. It consists mostly of the extracellular matrix proteins and a small number of highly differentiated chondrocytes. At present, various techniques for repairing joint surfaces damage, for example, the use of modified cell cultures and biodegradable scaffolds, are under investigation. Molecular mechanisms of cartilage tissue proliferation have been also actively studied in recent years. TGFß3, which plays a critical role in the proliferation of normal cartilage tissue, is one of the most important protein among cytokines and growth factors affecting chondrogenesis. By interacting directly with receptors on the cell membrane surface, TGFß3 triggers a cascade of molecular interactions involving transcription factor Sox9. In this review, we describe the effects of TGFß3 on the receptor complex activation and subsequent intracellular trafficking of Smad proteins and analyze the relation between these processes and upregulation of expression of major extracellular matrix genes, such as col2a1 and acan.

Biologically Regulated Marrow Stimulation by Blocking TGF-ß1 With Losartan Oral Administration Results in Hyaline-like Cartilage Repair: A Rabbit Osteochondral Defect Model.

BACKGROUND: Microfracture or bone marrow stimulation (BMS) is often the first choice for clinical treatment of cartilage injuries; however, fibrocartilage, not pure hyaline cartilage, has been reported because of the development of fibrosis in the repair tissue. Transforming growth factor ß1 (TGF-ß1), which can promote fibrosis, can be inhibited by losartan and potentially be used to reduce fibrocartilage. HYPOTHESIS: Blocking TGF-ß1 would improve cartilage healing in a rabbit knee BMS model via decreasing the amount of fibrocartilage and increasing hyaline-like cartilage formation. STUDY DESIGN: Controlled laboratory study. METHODS: An osteochondral defect was made in the patellar groove of 48 New Zealand White rabbits. The rabbits were divided into 3 groups: a defect group (defect only), a BMS group (osteochondral defect + BMS), and a BMS + losartan group (osteochondral defect + BMS + losartan). For the rabbits in the BMS + losartan group, losartan was administrated orally from the day after surgery through the day of euthanasia. Rabbits were sacrificed 6 or 12 weeks postoperatively. Macroscopic appearance, microcomputed tomography, histological assessment, and TGF-ß1 signaling pathway were evaluated at 6 and 12 weeks postoperatively. RESULTS: The macroscopic assessment of the repair revealed that the BMS + losartan group was superior to the other groups tested. Microcomputed tomography showed superior healing of the bony defect in the BMS + losartan group in comparison with the other groups. Histologically, fibrosis in the repair tissue of the BMS + losartan group was significantly reduced when compared with the other groups. Results obtained with the modified O'Driscoll International Cartilage Repair Society grading system yielded significantly superior scores in the BMS + losartan group as compared with both the defect group and the BMS group (F value: 15.8, P < .001, P = .012, respectively). TGF-ß1 signaling and TGF-ß-activated kinase 1 of the BMS + losartan group were significantly suppressed in the synovial tissues. CONCLUSION: By blocking TGF-ß1 with losartan, the repair cartilage tissue after BMS was superior to the other groups and consisted primarily of hyaline cartilage. These results should be easily translated to the clinic because losartan is a Food and Drug Administration-approved drug and it can be combined with the BMS technique for optimal repair of chondral defects. CLINICAL RELEVANCE: Biologically regulated marrow stimulation by blocking TGF-ß1 (oral intake of losartan) provides superior repair via decreasing fibrocartilage formation and resulting in hyaline-like cartilage as compared with outcomes from BMS only.

Cytokine Directed Chondroblast Trans-Differentiation: JAK Inhibition Facilitates Direct Reprogramming of Fibroblasts to Chondroblasts.

Osteoarthritis (OA) is a degenerative disease of the hyaline articular cartilage. This disease is progressive and may lead to disability. Researchers proposed many regenerative approaches to treat osteoarthritis, including stem cells. Trans-differentiation of a fully differentiated cell state directly into another different differentiated cell state avoids the disadvantages of fully reprogramming cells to induced pluripotent stem cells (iPSCs) in terms of faster reprogramming of the needed cells. Trans-differentiation also reduces the risk of tumor formation by avoiding the iPSC state. OSKM factors (Oct4, Sox2, Klf4, and cMyc) accompanied by the JAK-STAT pathway inhibition, followed by the introduction of specific differentiation factors, directly reprogrammed mouse embryonic fibroblasts to chondroblasts. Our results showed the absence of intermediate induced pluripotent stem cell formation. The resulting aggregates showed clear hyaline and hypertrophic cartilage. Tumor formation was absent in sub-cutaneous capsules transplanted in SCID mice.

3D bioprinted hydrogel model incorporating ß-tricalcium phosphate for calcified cartilage tissue engineering.

One promising strategy to reconstruct osteochondral defects relies on 3D bioprinted three-zonal structures comprised of hyaline cartilage, calcified cartilage, and subchondral bone. So far, several studies have pursued the regeneration of either hyaline cartilage or bone in vitro while-despite its key role in the osteochondral region-only few of them have targeted the calcified layer. In this work, we present a 3D biomimetic hydrogel scaffold containing ß-tricalcium phosphate (TCP) for engineering calcified cartilage through a co-axial needle system implemented in extrusion-based bioprinting process. After a thorough bioink optimization, we showed that 0.5% w/v TCP is the optimal concentration forming stable scaffolds with high shape fidelity and endowed with biological properties relevant for the development of calcified cartilage. In particular, we investigate the effect induced by ceramic nano-particles over the differentiation capacity of bioprinted bone marrow-derived human mesenchymal stem cells in hydrogel scaffolds cultured up to 21 d in chondrogenic media. To confirm the potential of the presented approach to generate a functional in vitro model of calcified cartilage tissue, we evaluated quantitatively gene expression of relevant chondrogenic (COL1, COL2, COL10A1, ACAN) and osteogenic (ALPL, BGLAP) gene markers by means of RT-qPCR and qualitatively by means of fluorescence immunocytochemistry.

Maternal HMB treatment affects bone and hyaline cartilage development in their weaned piglets via the leptin/osteoprotegerin system.

It has been demonstrated in animal studies that prenatal administration of ß-hydroxy-ß-methylbutyrate (HMB, metabolite of leucine) influences general growth and mechanical endurance of long bones in newborn offspring in sex-dependent manner. The present experiment was conducted to evaluate the effect of HMB treatment of pregnant sows on bone development in offspring at weaning. From 70th day until the 90th day of gestation, sows received either a basal diet (n = 12) or the same diet supplemented with HMB (n = 12) at the dose of 0.2 g/kg of body weight/day. Femora obtained from six males and females in each group weaned at the age of 35 days were examined. Maternal HMB treatment significantly enhanced body weight and changed bone morphology increasing femur mechanical strength in both sexes. Maternal HMB supplementation also elevated bone micro- and macroelement concentrations and enhanced content of proteoglycans in articular cartilage. Based on the obtained results, it can be concluded that maternal HMB supplementation in the mid-gestation period significantly accelerated bone development in both sexes by upregulation of a multifactorial system including leptin and osteoprotegerin. However, the sex (irrespective of the HMB treatment) was the factor which influenced the collagen structure in cartilages and trabecular bone, as demonstrated both by the Picrosirius red staining and performed analysis of thermal stability of collagenous tissues. The structural differences in collagen between males and females were presumably related to a different collagen maturity. No studies conducted so far provided a detailed morphological analysis of bone, articular cartilage, growth plate and the activities of the somatotropic and pituitary-gonadal axes, as well as leptin/osteoprotegerin system in weaned offspring prenatally treated with HMB. This study showed also the relationship between the maternal HMB treatment and bone osteometric and mechanical traits, hormones, and growth and bone turnover markers such as leptin, osteoprotegerin and insulin-like growth factor-1.

Spatially confined induction of endochondral ossification by functionalized hydrogels for ectopic engineering of osteochondral tissues.

Despite the various reported approaches to generate osteochondral composites by combination of different cell types and materials, engineering of templates with the capacity to autonomously and orderly develop into cartilage-bone bi-layered structures remains an open challenge. Here, we hypothesized that the embedding of cells inducible to endochondral ossification (i.e. bone marrow derived mesenchymal stromal cells, BMSCs) and of cells capable of robust and stable chondrogenesis (i.e. nasal chondrocytes, NCs) adjacent to each other in bi-layered hydrogels would develop directly in vivo into osteochondral tissues. Poly(ethylene glycol) (PEG) hydrogels were functionalized with TGFß3 or BMP-2, enzymatically polymerized encapsulating human BMSCs, combined with a hydrogel layer containing human NCs and ectopically implanted in nude mice without pre-culture. The BMSC-loaded layers reproducibly underwent endochondral ossification and generated ossicles containing bone and marrow. The NC-loaded layers formed cartilage tissues, which (under the influence of BMP-2 but not of TGFß3 from the neighbouring layer) remained phenotypically stable. The proposed strategy, resulting in orderly connected osteochondral composites, should be further assessed for the repair of osteoarticular defects and will be useful to model developmental processes leading to cartilage-bone interfaces.

Bone Marrow Aspirate Concentrate for Cartilage Defects of the Knee: From Bench to Bedside Evidence.

Objective To critically evaluate the current basic science, translational, and clinical data regarding bone marrow aspirate concentrate (BMAC) in the setting of focal cartilage defects of the knee and describe clinical indications and future research questions surrounding the clinical utility of BMAC for treatment of these lesions. Design A literature search was performed using the PubMed and Ovid MEDLINE databases for studies in English (1980-2017) using keywords, including ["bone marrow aspirate" and "cartilage"], ["mesenchymal stem cells" and "cartilage"], and ["bone marrow aspirate" and "mesenchymal stem cells" and "orthopedics"]. A total of 1832 articles were reviewed by 2 independent authors and additional literature found through scanning references of cited articles. Results BMAC has demonstrated promising results in the clinical application for repair of chondral defects as an adjuvant procedure or as an independent management technique. A subcomponent of BMAC, bone marrow derived-mesenchymal stem cells (MSCs) possess the ability to differentiate into cells important for osteogenesis and chondrogenesis. Modulation of paracrine signaling is perhaps the most important function of BM-MSCs in this setting. In an effort to increase the cellular yield, authors have shown the ability to expand BM-MSCs in culture while maintaining phenotype. Conclusions Translational studies have demonstrated good clinical efficacy of BMAC both concomitant with cartilage restoration procedures, at defined time points after surgery, and as isolated injections. Early clinical data suggests BMAC may help stimulate a more robust hyaline cartilage repair tissue response. Numerous questions remain regarding BMAC usage, including cell source, cell expansion, optimal pathology, and injection timing and quantity.

Effect of Dietary Phytase Supplementation on Bone and Hyaline Cartilage Development of Broilers Fed with Organically Complexed Copper in a Cu-Deficient Diet.

Tibial mechanical, chemical, and histomorphometrical traits were investigated for growing male Ross 308 broiler chickens fed diets that had copper (Cu) from organic source at a lowered level of 25% of the daily requirement (4 mg kg-1 of a premix) with or without phytase. Dietary treatments were control non-copper, non-phytase group (0 Suppl); 4 mg kg-1 Cu non-phytase group (25%Cu); and 4 mg kg-1 Cu + 500 FTU kg-1 phytase group (25%Cu + phyt). The results show that birds fed with the addition of phytase exhibited improved weight gain and final body weight and had increased serum IGF-1 and osteocalcin concentrations. The serum concentration of Cu and P did not differ between groups; however, Ca concentration decreased in the 25%Cu + phyt group when compared to the 25%Cu group. Added Cu increased bone Ca, P, Cu, and ash content in Cu-supplemented groups, but bone weight and length increased only by the addition of phytase. Bone geometry, yield, and ultimate strengths were affected by Cu and phytase addition. A decrease of the elastic stress and ultimate stress of the tibia in Cu-supplemented groups was observed. The histomorphometric analysis showed a positive effect of Cu supplementation on real bone volume and trabecular thickness in the tibia metaphyseal trabeculae; additionally, phytase increased the trabeculea number. The supplementation with Cu significantly increased the total articular cartilage and growth plate cartilage thickness; however, the changes in thickness of particular zones were dependent upon phytase addition. In summary, dietary Cu supplements given to growing broilers with Cu in their diet restricted to 25% of the daily requirement had a positive effect on bone metabolism, and phytase supplementation additionally improved cartilage development.

Vascular Endothelial Growth Factor Sequestration Enhances In Vivo Cartilage Formation.

Autologous chondrocyte transplantation for cartilage repair still has unsatisfactory clinical outcomes because of inter-donor variability and poor cartilage quality formation. Re-differentiation of monolayer-expanded human chondrocytes is not easy in the absence of potent morphogens. The Vascular Endothelial Growth Factor (VEGF) plays a master role in angiogenesis and in negatively regulating cartilage growth by stimulating vascular invasion and ossification. Therefore, we hypothesized that its sole microenvironmental blockade by either VEGF sequestration by soluble VEGF receptor-2 (Flk-1) or by antiangiogenic hyperbranched peptides could improve chondrogenesis of expanded human nasal chondrocytes (NC) freshly seeded on collagen scaffolds. Chondrogenesis of several NC donors was assessed either in vitro or ectopically in nude mice. VEGF blockade appeared not to affect NC in vitro differentiation, whereas it efficiently inhibited blood vessel ingrowth in vivo. After 8 weeks, in vivo glycosaminoglycan deposition was approximately two-fold higher when antiangiogenic approaches were used, as compared to the control group. Our data indicates that the inhibition of VEGF signaling, independently of the specific implementation mode, has profound effects on in vivo NC chondrogenesis, even in the absence of chondroinductive signals during prior culture or at the implantation site.

Sprifermin (rhFGF18) enables proliferation of chondrocytes producing a hyaline cartilage matrix.

OBJECTIVE: Fibroblast growth factor (FGF) 18 has been shown to increase cartilage volume when injected intra-articularly in animal models of osteoarthritis (OA) and in patients with knee OA (during clinical development of the recombinant human FGF18, sprifermin). However, the exact nature of this effect is still unknown. In this study, we aimed to investigate the effects of sprifermin at the cellular level. DESIGN: A combination of different chondrocyte culture systems was used and the effects of sprifermin on proliferation, the phenotype and matrix production were evaluated. The involvement of MAPKs in sprifermin signalling was also studied. RESULTS: In monolayer, we observed that sprifermin promoted a round cell morphology and stimulated both cellular proliferation and Sox9 expression while strongly decreasing type I collagen expression. In 3D culture, sprifermin increased the number of matrix-producing chondrocytes, improved the type II:I collagen ratio and enabled human OA chondrocytes to produce a hyaline extracellular matrix (ECM). Furthermore, we found that sprifermin displayed a 'hit and run' mode of action, with intermittent exposure required for the compound to fully exert its anabolic effect. Finally, sprifermin appeared to signal through activation of ERK. CONCLUSIONS: Our results indicate that intermittent exposure to sprifermin leads to expansion of hyaline cartilage-producing chondrocytes. These in vitro findings are consistent with the increased cartilage volume observed in the knees of OA patients after intra-articular injection with sprifermin in clinical studies.

Regeneration of hyaline-like cartilage in situ with SOX9 stimulation of bone marrow-derived mesenchymal stem cells.

Microfracture, a common procedure for treatment of cartilage injury, induces fibrocartilage repair by recruiting bone marrow derived mesenchymal stem cells (MSC) to the site of cartilage injury. However, fibrocartilage is inferior biomechanically to hyaline cartilage. SRY-type high-mobility group box-9 (SOX9) is a master regulator of chondrogenesis by promoting proliferation and differentiation of MSC into chondrocytes. In this study we aimed to test the therapeutic potential of cell penetrating recombinant SOX9 protein in regeneration of hyaline cartilage in situ at the site of cartilage injury. We generated a recombinant SOX9 protein which was fused with super positively charged green fluorescence protein (GFP) (scSOX9) to facilitate cell penetration. scSOX9 was able to induce chondrogenesis of bone marrow derived MSC in vitro. In a rabbit cartilage injury model, scSOX9 in combination with microfracture significantly improved quality of repaired cartilage as shown by macroscopic appearance. Histological analysis revealed that the reparative tissue induced by microfracture with scSOX9 had features of hyaline cartilage; and collagen type II to type I ratio was similar to that in normal cartilage. This short term in vivo study demonstrated that when administered at the site of microfracture, scSOX9 was able to induce reparative tissue with features of hyaline cartilage.

Sol-gel derived lithium-releasing glass for cartilage regeneration.

Wnt-signalling cascade is one of the crucial pathways involved in the development and homeostasis of cartilage. Influencing this pathway can potentially contribute to improved cartilage repair or regeneration. One key molecular regulator of the Wnt pathway is the glycogen synthase kinase-3 enzyme, the inhibition of which allows initiation of the signalling pathway. This study aims to utilise a binary SiO2-Li2O sol-gel derived glass for controlled delivery of lithium, a known glycogen synthase kinase-3 antagonist. The effect of the dissolution products of the glass on chondrogenic differentiation in an in vitro 3D pellet culture model is reported. Dissolution products that contained 5 mM lithium and 3.5 mM silicon were capable of inducing chondrogenic differentiation and hyaline cartilaginous matrix formation without the presence of growth factors such as TGF-ß3. The results suggest that sol-gel derived glass has the potential to be used as a delivery vehicle for therapeutic lithium ions in cartilage regeneration applications.

First and second order stereology of hyaline cartilage: Application on mice femoral cartilage.

Stereological techniques could be considered in research on cartilage to obtain quantitative data. The present study aimed to explain application of the first- and second-order stereological methods on articular cartilage of mice and the methods applied on the mice exposed to cadmium (Cd). The distal femoral articular cartilage of BALB/c mice (control and Cd-treated) was removed. Then, volume and surface area of the cartilage and number of chondrocytes were estimated using Cavalieri and optical dissector techniques on isotropic uniform random sections. Pair-correlation function [g(r)] and cross-correlation function were calculated to express the spatial arrangement of chondrocytes-chondrocytes and chondrocytes-matrix (chondrocyte clustering/dispersing), respectively. The mean±standard deviation of the cartilage volume, surface area, and thickness were 1.4±0.1mm3, 26.2±5.4mm2, and 52.8±6.7µm, respectively. Besides, the mean number of chondrocytes was 680±200 (×103). The cartilage volume, cartilage surface area, and number of chondrocytes were respectively reduced by 25%, 27%, and 27% in the Cd-treated mice in comparison to the control animals (p<0.03). Estimates of g(r) for the cells and matrix against the dipole distances, r, have been plotted. This plot showed that the chondrocytes and the matrix were neither dispersed nor clustered in the two study groups. Application of design-based stereological methods and also evaluation of spatial arrangement of the cartilage components carried potential advantages for investigating the cartilage in different joint conditions. Chondrocyte clustering/dispersing and cellularity can be evaluated in cartilage assessment in normal or abnormal situations.

Possible role of the Ec peptide of IGF­1Ec in cartilage repair.

The Ec peptide (PEc) of insulin-like growth factor 1 Ec (IGF-1Ec) induces human mesenchymal stem cell (hMSC) mobilization and activates extracellular signal­regulated kinase 1/2 (ERK 1/2) in various cells. The aim of the present study was to examine the effects of PEc on the mobilization and differentiation of hMSCs, as well as the possibility of its implementation in combination with transforming growth factor ß1 (TGF­ß1) for cartilage repair. The effects of the exogenous administration of PEc and TGF­ß1, alone and in combination, on hMSCs were assessed using a trypan blue assay, reverse transcription-quantitative polymerase chain reaction, western blot analysis, Alcian blue staining, wound healing assays and migration/invasion assays. It was determined that PEc is involved in the differentiation process of hMSCs towards hyaline cartilage. Treatment of hMSCs with either PEc, TGF­ß1 or both, demonstrated comparable cartilage matrix deposition. Furthermore, treatment with PEc in combination with TGF­ß1 was associated with a significant increase in hMSC mobilization when compared with treatment with TGF­ß1 or PEc alone (P<0.05). Thus, PEc appears to facilitate in vitro hMSC mobilization and differentiation towards chondrocytes, enhancing the role of TGF­ß1.

Intra-articular administration of hyaluronic acid increases the volume of the hyaline cartilage regenerated in a large osteochondral defect by implantation of a double-network gel.

Implantation of PAMPS/PDMAAm double-network (DN) gel can induce hyaline cartilage regeneration in the osteochondral defect. However, it is a problem that the volume of the regenerated cartilage tissue is gradually reduced at 12 weeks. This study investigated whether intra-articular administration of hyaluronic acid (HA) increases the volume of the cartilage regenerated with the DN gel at 12 weeks. A total of 48 rabbits were used in this study. A cylindrical osteochondral defect created in the bilateral femoral trochlea was treated with DN gel (Group DN) or left without any implantation (Group C). In both Groups, we injected 1.0 mL of HA in the left knee, and 1.0 mL of saline solution in the right knee. Quantitative histological evaluations were performed at 2, 4, and 12 weeks, and PCR analysis was performed at 2 and 4 weeks after surgery. In Group DN, the proteoglycan-rich area was significantly greater in the HA-injected knees than in the saline-injected knees at 12 weeks (P = 0.0247), and expression of type 2 collagen, aggrecan, and Sox9 mRNAs was significantly greater in the HA-injected knees than in the saline-injected knees at 2 weeks (P = 0.0475, P = 0.0257, P = 0.0222, respectively). The intra-articular administration of HA significantly enhanced these gene expression at 2 weeks and significantly increased the volume of the hyaline cartilage regenerated by implantation of a DN gel at 12 weeks. This information is important to develop an additional method to increase the volume of the hyaline cartilage tissue in a potential cartilage regeneration strategy using the DN gel.

Similar hyaline-like cartilage repair of osteochondral defects in rabbits using isotropic and anisotropic collagen scaffolds.

Lesions in knee joint articular cartilage (AC) have limited repair capacity. Many clinically available treatments induce a fibrous-like cartilage repair instead of hyaline cartilage. To induce hyaline cartilage repair, we hypothesized that type I collagen scaffolds with fibers aligned perpendicular to the AC surface would result in qualitatively better tissue repair due to a guided cellular influx from the subchondral bone. By specific freezing protocols, type I collagen scaffolds with isotropic and anisotropic fiber architectures were produced. Rabbits were operated on bilaterally and two full thickness defects were created in each knee joint. The defects were filled with (1) an isotropic scaffold, (2) an anisotropic scaffold with pores parallel to the cartilage surface, and (3) an anisotropic scaffold with pores perpendicular to the cartilage surface. Empty defects served as controls. After 4 (n=13) and 12 (n=13) weeks, regeneration was scored qualitatively and quantitatively using histological analysis and a modified O'Driscoll score. After 4 weeks, all defects were completely filled with partially differentiated hyaline cartilage tissue. No differences in O'Driscoll scores were measured between empty defects and scaffold types. After 12 weeks, all treatments led to hyaline cartilage repair visualized by increased glycosaminoglycan staining. Total scores were significantly increased for parallel anisotropic and empty defects over time (p<0.05). The results indicate that collagen scaffolds allow the formation of hyaline-like cartilage repair. Fiber architecture had no effect on cartilage repair.

Evaluation of propargyl alcohol toxicity and carcinogenicity in F344/N rats and B6C3F1/N mice following whole-body inhalation exposure.

Propargyl alcohol (PA) is a high production volume chemical used in synthesis of many industrial chemicals and agricultural products. Despite the potential for prolonged or accidental exposure to PA in industrial settings, the toxicity potential of PA was not well characterized. To address the knowledge gaps relevant to the toxicity profile of PA, the National Toxicology Program (NTP) conducted 2-week, 14-week and 2-year studies in male and female F344/N rats and B6C3F1/N mice. For the 2-week inhalation study, the rats and mice were exposed to 0, 31.3, 62.5, 125, 250 or 500ppm. Significant mortality was observed in both rats and mice exposed to ≥125ppm of PA. The major target organ of toxicity in both mice and rats was the liver with exposure-related histopathological changes (250 and 500ppm). Based on the decreased survival in the 2-week study, the rats and mice were exposed to 0, 4, 8, 16, 32 or 64ppm of PA in the 14-week study. No treatment-related mortality was observed. Mean body weights of male (≥8ppm) and female mice (32 and 64ppm) were significantly decreased (7-16%). Histopathological changes were noted in the nasal cavity, and included suppurative inflammation, squamous metaplasia, hyaline droplet accumulation, olfactory epithelium atrophy, and necrosis. In the 2-year inhalation studies, the rats were exposed to 0, 16, 32 and 64ppm of PA and the mice were exposed to 0, 8, 16 and 32ppm of PA. Survival of male rats was significantly reduced (32 and 64ppm). Mean body weights of 64ppm male rats were significantly decreased relative to the controls. Both mice and rats showed a spectrum of non-neoplastic changes in the nose. Increased neoplastic incidences of nasal respiratory/transitional epithelial adenoma were observed in both rats and mice. The incidence of mononuclear cell leukemia was significantly increased in male rats and was considered to be treatment-related. In conclusion, the key findings from this study indicated that the nose was the primary target organ of toxicity for PA. Long term inhalation exposure to PA led to nonneoplastic changes in the nose, and increased incidences of respiratory/transitional epithelial adenomas in both mice and rats. Increased incidences of harderian gland adenoma may also have been related to exposure to PA in male mice.

Influence of the gel thickness on in vivo hyaline cartilage regeneration induced by double-network gel implanted at the bottom of a large osteochondral defect: short-term results.

BACKGROUND: A double-network (DN) gel, which is composed of poly(2-acrylamido-2-methylpropanesulfonic acid) and poly(N,N'-dimethyl acrylamide), can induce hyaline cartilage regeneration in vivo in a large osteochondral defect. The purpose of this study was to clarify the influence of the thickness of the implanted DN gel on the induction ability of hyaline cartilage regeneration. METHODS: Thirty-eight mature rabbits were used in this study. We created an osteochondral defect having a diameter of 4.3-mm in the patellofemoral joint. The knees were randomly divided into 4 groups (Group I: 0.5-mm thick gel, Group II: 1.0-mm thick gel, Group III: 5.0-mm thick gel, and Group IV: untreated control). Animals in each group were further divided into 3 sub-groups depending on the gel implant position (2.0-, 3.0-, or 4.0-mm depth from the articular surface) in the defect. The regenerated tissues were evaluated with the Wayne's gross and histological grading scales and real time PCR analysis of the cartilage marker genes at 4 weeks. RESULTS: According to the total Wayne's score, when the depth of the final vacant space was set at 2.0 mm, the scores in Groups I, II, and III were significantly greater than that Group IV (p<0.05), although there were no significant differences between Groups I and IV at a 3.0-mm deep vacant space. The expression levels of type-2 collagen in Groups II and III were significantly higher (p<0.05) than that in Group IV. CONCLUSIONS: The 1.0-mm thick DN gel sheet had the same ability to induce hyaline cartilage regeneration as the 5.0-mm thick DN gel plug. However, the induction ability of the 0.5-mm thick sheet was significantly lower when compared with the 1.0-mm thick gel sheet. The 1.0-mm DN gel sheet is a promising device to establish a cell-free cartilage regeneration strategy that minimizes bone loss from the gel implantation.

Effect of excessive methionine on the development of the cranial growth plate in newborn rats.

OBJECTIVE: Methionine is an essential amino acid and pivotal for normal growth and development. However, previous animal studies have shown that excessive maternal intake of methionine causes growth restrictions, organ damages, and abnormal growth of the mandible in newborn animals. However, the effect of excessive methionine on the development of the cranial growth plate is unknown. This study investigated histological alterations of the cranial growth plate induced by high methionine administration in newborn rats. DESIGN: Twenty pregnant dams were divided into a control and an experimental group. The controls received a diet for rats and the experimental group was fed from the 18th gestational day with a special manufactured high methionine diet for rats. The high methionine diet was maintained until the end of the lactation phase (day 20). The offspring of both groups were killed at day 10 or 20 postnatally and their spheno-occipital synchondroses were collected for histological analysis. RESULTS: The weight of the high-dose methionine treated experimental group was considerably reduced in comparison to the control group at day 10 and 20 postnatally. The cartilaginous area of the growth plate and the height of the proliferative zone were markedly reduced at postnatal day 10 in the experimental group. CONCLUSIONS: In summary, the diet-induced hypermethioninemia in rat dams resulted in growth retardations and histomorphological changes of the spheno-occipital synchondrosis, an important craniofacial growth centre in newborns. This finding may elucidate facial dysmorphoses reported in patients suffering from hypermethioninemia.

Hyaline cartilage regeneration by combined therapy of microfracture and long-term bone morphogenetic protein-2 delivery.

Microfracture of cartilage induces migration of bone-marrow-derived mesenchymal stem cells. However, this treatment often results in fibrocartilage regeneration. Growth factors such as bone morphogenetic protein (BMP)-2 induce the differentiation of bone-marrow-derived mesenchymal stem cells into chondrocytes, which can be used for hyaline cartilage regeneration. Here, we tested the hypothesis that long-term delivery of BMP-2 to cartilage defects subjected to microfracture results in regeneration of high-quality hyaline-like cartilage, as opposed to short-term delivery of BMP-2 or no BMP-2 delivery. Heparin-conjugated fibrin (HCF) and normal fibrin were used as carriers for the long- and short-term delivery of BMP-2, respectively. Rabbit articular cartilage defects were treated with microfracture combined with one of the following: no treatment, fibrin, short-term delivery of BMP-2, HCF, or long-term delivery of BMP-2. Eight weeks after treatment, histological analysis revealed that the long-term delivery of BMP-2 group (microfracture + HCF + BMP-2) showed the most staining with alcian blue. A biochemical assay, real-time polymerase chain reaction assay and Western blot analysis all revealed that the long-term delivery of BMP-2 group had the highest glucosaminoglycan content as well as the highest expression level of collagen type II. Taken together, the long-term delivery of BMP-2 to cartilage defects subjected to microfracture resulted in regeneration of hyaline-like cartilage, as opposed to short-term delivery or no BMP-2 delivery. Therefore, this method could be more convenient for hyaline cartilage regeneration than autologous chondrocyte implantation due to its less invasive nature and lack of cell implantation.