Cell-nonautonomous local and systemic responses to cell arrest enable long-bone catch-up growth in developing mice.

Catch-up growth after insults to growing organs is paramount to achieving robust body proportions. In fly larvae, injury to individual tissues is followed by local and systemic compensatory mechanisms that allow the damaged tissue to regain normal proportions with other tissues. In vertebrates, local catch-up growth has been described after transient reduction of bone growth, but the underlying cellular responses are controversial. We developed an approach to study catch-up growth in foetal mice in which mosaic expression of the cell cycle suppressor p21 is induced in the cartilage cells (chondrocytes) that drive long-bone elongation. By specifically targeting p21 expression to left hindlimb chondrocytes, the right limb serves as an internal control. Unexpectedly, left-right limb symmetry remained normal, revealing deployment of compensatory mechanisms. Above a certain threshold of insult, an orchestrated response was triggered involving local enhancement of bone growth and systemic growth reduction that ensured that body proportions were maintained. The local response entailed hyperproliferation of spared left limb chondrocytes that was associated with reduced chondrocyte density. The systemic effect involved impaired placental function and IGF signalling, revealing bone-placenta communication. Therefore, vertebrates, like invertebrates, can mount coordinated local and systemic responses to developmental insults that ensure that normal body proportions are maintained.

Analysis of short-term treatment with the phosphodiesterase type 5 inhibitor tadalafil on long bone development in young rats.

Cyclic GMP (cGMP) is an important intracellular regulator of endochondral bone growth and skeletal remodeling. Tadalafil, an inhibitor of the phosphodiesterase (PDE) type 5 (PDE5) that specifically hydrolyzes cGMP, is increasingly used to treat children with pulmonary arterial hypertension (PAH), but the effect of tadalafil on bone growth and strength has not been previously investigated. In this study, we first analyzed the expression of transcripts encoding PDEs in primary cultures of chondrocytes from newborn rat epiphyses. We detected robust expression of PDE5 as the major phosphodiesterase hydrolyzing cGMP. Time-course experiments showed that C-type natriuretic peptide increased intracellular levels of cGMP in primary chondrocytes with a peak at 2 min, and in the presence of tadalafil the peak level of intracellular cGMP was 37% greater ( P < 0.01) and the decline was significantly attenuated. Next, we treated 1-mo-old Sprague Dawley rats with vehicle or tadalafil for 3 wk. Although 10 mg·kg-1·day-1 tadalafil led to a significant 52% ( P < 0.01) increase in tissue levels of cGMP and a 9% reduction ( P < 0.01) in bodyweight gain, it did not alter long bone length, cortical or trabecular bone properties, and histological features. In conclusion, our results indicate that PDE5 is highly expressed in growth plate chondrocytes, and short-term tadalafil treatment of growing rats at doses comparable to those used in children with PAH has neither obvious beneficial effect on long bone growth nor any observable adverse effect on growth plate structure and trabecular and cortical bone structure.

Specific Deletion of ß-Catenin in Col2-Expressing Cells Leads to Defects in Epiphyseal Bone.

The role of canonical Wnt/ß-catenin signaling in postnatal bone growth has not been fully defined. In the present studies, we generated ß-catenin conditional knockout (KO) mice and deleted ß-catenin in Col2-expressing chondrocytes and mesenchymal progenitor cells. Findings from analyzing the ß-cateninCol2CreER KO mice revealed severe bone destruction and bone loss phenotype in epiphyseal bone, probably due to the increase in osteoclast formation and the accumulation of adipocytes in this area. In addition, we also found bone destruction and bone loss phenotype in vertebral bone in ß-cateninCol2CreER KO mice. These findings indicate that ß-catenin signaling plays a critical role in postnatal bone remodeling. Our study provides new insights into the regulation of epiphyseal bone homeostasis at postnatal stage.

The effect of mechanical stretch stress on the differentiation and apoptosis of human growth plate chondrocytes.

The study is aimed to investigate the effect of stretch stress with different intensities on the differentiation and apoptosis of human plate chondrocytes. In the present study, the human epiphyseal plate chondrocytes were isolated and cultured in vitro. Toluidine blue staining and type II collagen immunohistochemical staining were used to identify the chondrocytes. Mechanical stretch stresses with different intensities were applied to intervene cells at 0-, 2000-, and 4000-µ strain for 6 h via a four-point bending system. The expression levels of COL2, COL10, Bax, Bcl-2, and PTHrp were detected by quantitative RT-PCR. Under the intervention of 2000-µ strain, the expression levels of COL2, COL10, and PTHrp increased significantly compared with the control group (P < 0.05), and the expression level of PCNA was also increased, but the difference was not statistically significant (P > 0.05). Under 4000-µ strain, however, the expression levels of PCNA, COL2, and PTHrp decreased significantly compared with the control group (P < 0.05), and the expression level of COL10 decreased slightly (P > 0.05). The ratio of Bcl-2/Bax gradually increased with the increase of stimulus intensity; both of the differences were detected to be statistically significant (P < 0.05). In conclusion, the apoptosis of growth plate chondrocytes is regulated by mechanical stretch stress. Appropriate stretch stress can effectively promote the cells' proliferation and differentiation, while excessive stretch stress inhibits the cells' proliferation and differentiation, even promotes their apoptosis. PTHrp may play an important role in this process.

7T MRI of distal radius trabecular bone microarchitecture: How trabecular bone quality varies depending on distance from end-of-bone.

PURPOSE: To use 7T magnetic resonance imaging (MRI) to determine how trabecular bone microarchitecture varies at the epiphysis, metaphysis, and diaphysis of the distal radius. MATERIALS AND METHODS: The distal radius of 24 females (mean age = 56 years, range = 24-78 years) was scanned on a 7T MRI using a 3D fast low-angle shot sequence (0.169 × 0.169 × 1 mm). Digital topological analysis was applied at the epiphysis, metaphysis, and diaphysis to compute: total trabecular bone volume; trabecular thickness, number, connectivity, and erosion index (a measure of network resorption). Differences and correlations were assessed using standard statistical methods. RESULTS: The metaphysis and epiphysis had 83-123% greater total bone volume and 14-16% greater trabecular number than the diaphysis (both P < 0.0001). The erosion index was significantly higher at the diaphysis than the metaphysis and epiphysis (both P < 0.01). The most elderly volunteers had lower trabecular number (<66 years mean 0.29 ± 0.01; ≥66 years, 0.27 ± 0.02, P < 0.05) and higher erosion index (<66 years mean 1.18 ± 0.17; age ≥66 years, mean 1.42 ± 0.46, P < 0.05) at the epiphysis; differences not detected by total trabecular bone volume. CONCLUSION: 7T MRI reveals trabecular bone microarchitecture varies depending on scan location at the end-of-bone, being of overall higher quality distally (epiphysis) than proximally (diaphysis). Age-related differences in trabecular microarchitecture can be detected by 7T MRI. The results highlight the potential sensitivity of 7T MRI to microarchitectural differences and the potential importance of standardizing scan location for future clinical studies of fracture risk or treatment response. LEVEL OF EVIDENCE: 3 J. Magn. Reson. Imaging 2017;45:872-878.

Second harmonic generation imaging reveals a distinct organization of collagen fibrils in locations associated with cartilage growth.

PURPOSE: The articular-epiphyseal cartilage complex (AECC) is responsible for the expansion of the bone ends and serves the function of the articular cartilage in juvenile mammals. Bundles of collagen fibrils surrounding cells were in the literature observed more frequently near the articular surface of the AECC. The articular surface, the perichondrium, and cartilage canals are interfaces where appositional growth of the AECC has been demonstrated. The current study aimed to evaluate the potential of second harmonic generation (SHG) to locate the collagen fibril bundles near the articular surface and to examine whether a comparable collagen fibril organization could be observed near the other interfaces of the AECC. MATERIALS AND METHODS: The study included the femoral condyle of four piglets aged 82-141 days. The forward and backward scattered SHG, and their ratio, was analyzed across the AECC using objectives with different numerical aperture. Two-photon-excited fluorescence was used to visualize cells. RESULTS: A similar pattern of collagen fibril organization was observed near the articular surface, around cartilage canals, and adjacent to the perichondrium. The pattern consisted of a higher ratio of forward to backward scattered SHG that increased relative to the surrounding matrix at lower numerical aperture. This was interpreted to reflect collagen fibril bundles in the territorial matrix of cells in these areas. CONCLUSIONS: The observed arrangement of collagen fibrils was suggested to be related to the presumed different growth activity in these areas and indicated that SHG may be used as an indirect and label-free marker for cartilage matrix growth.

Regionally-derived cell populations and skeletal stem cells from human foetal femora exhibit specific osteochondral and multi-lineage differentiation capacity in vitro and ex vivo.

BACKGROUND: Adult skeletal stem cells (SSCs) often exhibit limited in vitro expansion with undesirable phenotypic changes and loss of differentiation capacity. Foetal tissues offer an alternative cell source, providing SSCs which exhibit desirable differentiation capacity over prolonged periods, ideal for extensive in vitro and ex vivo investigation of fundamental bone biology and skeletal development. METHODS: We have examined the derivation of distinct cell populations from human foetal femora. Regionally isolated populations including epiphyseal and diaphyseal cells were carefully dissected. Expression of the SSC marker Stro-1 was also found in human foetal femora over a range of developmental stages and subsequently utilised for immuno-selection. RESULTS: Regional populations exhibited chondrogenic (epiphyseal) and osteogenic (diaphyseal) phenotypes following in vitro and ex vivo characterisation and molecular analysis, indicative of native SSC maturation during skeletal development. However, each population exhibited potential for induced multi-lineage differentiation towards bone (bone nodule formation), cartilage (proteoglycan and mucopolysaccharide deposition) and fat (lipid deposition), suggesting the presence of a shared stem cell sub-population. This shared sub-population may be comprised of Stro-1+ cells, which were later identified and immuno-selected from whole foetal femora exhibiting multi-lineage differentiation capacity in vitro and ex vivo. CONCLUSIONS: Distinct populations were isolated from human foetal femora expressing osteochondral differentiation capacity. Stro-1 immuno-selected SSCs were isolated from whole femora expressing desirable multi-lineage differentiation capacity over prolonged in vitro expansion, superior to their adult-derived counterparts, providing a valuable cell source with which to study bone biology and skeletal development.

Delayed hypertrophic differentiation of epiphyseal chondrocytes contributes to failed secondary ossification in mucopolysaccharidosis VII dogs.

Mucopolysaccharidosis (MPS) VII is a lysosomal storage disorder characterized by deficient ß-glucuronidase activity, which leads to the accumulation of incompletely degraded glycosaminoglycans (GAGs). MPS VII patients present with severe skeletal abnormalities, which are particularly prevalent in the spine. Incomplete cartilage-to-bone conversion in MPS VII vertebrae during postnatal development is associated with progressive spinal deformity and spinal cord compression. The objectives of this study were to determine the earliest postnatal developmental stage at which vertebral bone disease manifests in MPS VII and to identify the underlying cellular basis of impaired cartilage-to-bone conversion, using the naturally-occurring canine model. Control and MPS VII dogs were euthanized at 9 and 14 days-of-age, and vertebral secondary ossification centers analyzed using micro-computed tomography, histology, qPCR, and protein immunoblotting. Imaging studies and mRNA analysis of bone formation markers established that secondary ossification commences between 9 and 14 days in control animals, but not in MPS VII animals. mRNA analysis of differentiation markers revealed that MPS VII epiphyseal chondrocytes are unable to successfully transition from proliferation to hypertrophy during this critical developmental window. Immunoblotting demonstrated abnormal persistence of Sox9 protein in MPS VII cells between 9 and 14 days-of-age, and biochemical assays revealed abnormally high intra and extracellular GAG content in MPS VII epiphyseal cartilage at as early as 9 days-of-age. In contrast, assessment of vertebral growth plates and primary ossification centers revealed no significant abnormalities at either age. The results of this study establish that failed vertebral bone formation in MPS VII can be traced to the failure of epiphyseal chondrocytes to undergo hypertrophic differentiation at the appropriate developmental stage, and suggest that aberrant processing of Sox9 protein may contribute to this cellular dysfunction. These results also highlight the importance of early diagnosis and therapeutic intervention to prevent the progression of debilitating skeletal disease in MPS patients.

The canine epiphyseal-derived mesenchymal stem cells are comparable to bone marrow derived-mesenchymal stem cells.

Mesenchymal stem cells (MSCs) hold great potential in cell therapy and have attracted increasing interests in a wide range of biomedical sciences. However, the scarcity of MSCs and the prolonged isolation procedure limited the clinical application. To address these 2 issues, we developed a method to isolate MSCs from bone biopsy tissues of euthanized canine body donors. Compared to the traditional method to isolate MSCs from aspirated bone marrow (BMSCs), the isolation procedure for MSCs from harvested epiphyseal cancellous bone (EMSCs) was less time-consuming. The isolated EMSCs had similar plastic-adherence, tri-lineage differentiation and consistent surface marker profiles compared to BMSCs. We harvested BMSCs and EMSCs from 24 euthanized cases from clinics and 42 euthanized donors from a local shelter. The successful rate for EMSC isolation is significantly higher compared to BMSC isolation, while the other properties of the isolated MSCs including the clonogenicity, proliferative potentials and molecular phenotypes were not discernibly different between the MSCs established by the two methods. In conclusion, we demonstrated a new procedure to harvest MSCs by bone biopsy at the epiphyseal region. This method is less time consuming and more reliable, and the resulting MSCs are comparable to those harvested by bone marrow aspiration. The combination of the two methods can greatly improve the efficiency to harvest MSCs.

MicroRNA-146a regulates human foetal femur derived skeletal stem cell differentiation by down-regulating SMAD2 and SMAD3.

MicroRNAs (miRs) play a pivotal role in a variety of biological processes including stem cell differentiation and function. Human foetal femur derived skeletal stem cells (SSCs) display enhanced proliferation and multipotential capacity indicating excellent potential as candidates for tissue engineering applications. This study has examined the expression and role of miRs in human foetal femur derived SSC differentiation along chondrogenic and osteogenic lineages. Cells isolated from the epiphyseal region of the foetal femur expressed higher levels of genes associated with chondrogenesis while cells from the foetal femur diaphyseal region expressed higher levels of genes associated with osteogenic differentiation. In addition to the difference in osteogenic and chondrogenic gene expression, epiphyseal and diaphyseal cells displayed distinct miRs expression profiles. miR-146a was found to be expressed by human foetal femur diaphyseal cells at a significantly enhanced level compared to epiphyseal populations and was predicted to target various components of the TGF-ß pathway. Examination of miR-146a function in foetal femur cells confirmed regulation of protein translation of SMAD2 and SMAD3, important TGF-ß and activin ligands signal transducers following transient overexpression in epiphyseal cells. The down-regulation of SMAD2 and SMAD3 following overexpression of miR-146a resulted in an up-regulation of the osteogenesis related gene RUNX2 and down-regulation of the chondrogenesis related gene SOX9. The current findings indicate miR-146a plays an important role in skeletogenesis through attenuation of SMAD2 and SMAD3 function and provide further insight into the role of miRs in human skeletal stem cell differentiation modulation with implications therein for bone reparation.

Epiphyseal chondrocyte secondary ossification centers require thyroid hormone activation of Indian hedgehog and osterix signaling.

Thyroid hormones (THs) are known to regulate endochondral ossification during skeletal development via acting directly in chondrocytes and osteoblasts. In this study, we focused on TH effects on the secondary ossification center (SOC) because the time of appearance of SOCs in several species coincides with the time when peak levels of TH are attained. Accordingly, micro-computed tomography (µCT) evaluation of femurs and tibias at day 21 in TH-deficient and control mice revealed that endochondral ossification of SOCs is severely compromised owing to TH deficiency and that TH treatment for 10 days completely rescued this phenotype. Staining of cartilage and bone in the epiphysis revealed that whereas all of the cartilage is converted into bone in the prepubertal control mice, this conversion failed to occur in the TH-deficient mice. Immunohistochemistry studies revealed that TH treatment of thyroid stimulating hormone receptor mutant (Tshr(-/-) ) mice induced expression of Indian hedgehog (Ihh) and Osx in type 2 collagen (Col2)-expressing chondrocytes in the SOC at day 7, which subsequently differentiate into type 10 collagen (Col10)/osteocalcin-expressing chondro/osteoblasts at day 10. Consistent with these data, treatment of tibia cultures from 3-day-old mice with 10 ng/mL TH increased expression of Osx, Col10, alkaline phosphatase (ALP), and osteocalcin in the epiphysis by sixfold to 60-fold. Furthermore, knockdown of the TH-induced increase in Osx expression using lentiviral small hairpin RNA (shRNA) significantly blocked TH-induced ALP and osteocalcin expression in chondrocytes. Treatment of chondrogenic cells with an Ihh inhibitor abolished chondro/osteoblast differentiation and SOC formation. Our findings indicate that TH regulates the SOC initiation and progression via differentiating chondrocytes into bone matrix-producing osteoblasts by stimulating Ihh and Osx expression in chondrocytes.

Isolation and characterization of novel murine epiphysis derived mesenchymal stem cells.

BACKGROUND: While bone marrow (BM) is a rich source of mesenchymal stem cells (MSCs), previous studies have shown that MSCs derived from mouse BM (BMMSCs) were difficult to manipulate as compared to MSCs derived from other species. The objective of this study was to find an alternative murine MSCs source that could provide sufficient MSCs. METHODOLOGY/PRINCIPAL FINDINGS: In this study, we described a novel type of MSCs that migrates directly from the mouse epiphysis in culture. Epiphysis-derived MSCs (EMSCs) could be extensively expanded in plastic adherent culture, and they had a greater ability for clonogenic formation and cell proliferation than BMMSCs. Under specific induction conditions, EMSCs demonstrated multipotency through their ability to differentiate into adipocytes, osteocytes and chondrocytes. Immunophenotypic analysis demonstrated that EMSCs were positive for CD29, CD44, CD73, CD105, CD166, Sca-1 and SSEA-4, while negative for CD11b, CD31, CD34 and CD45. Notably, EMSCs did not express major histocompatibility complex class I (MHC I) or MHC II under our culture system. EMSCs also successfully suppressed the proliferation of splenocytes triggered by concanavalin A (Con A) or allogeneic splenocytes, and decreased the expression of IL-1, IL-6 and TNF-α in Con A-stimulated splenocytes suggesting their anti-inflammatory properties. Moreover, EMSCs enhanced fracture repair, ameliorated necrosis in ischemic skin flap, and improved blood perfusion in hindlimb ischemia in the in vivo experiments. CONCLUSIONS/SIGNIFICANCES: These results indicate that EMSCs, a new type of MSCs established by our simple isolation method, are a preferable alternative for mice MSCs due to their better growth and differentiation potentialities.

Role of tartrate-resistant acid phosphatase (TRAP) in long bone development.

Tartrate resistant acid phosphatase (TRAP) was shown to be critical for skeleton development, and TRAP deficiency leads to a reduced resorptive activity during endochondral ossification resulting in an osteopetrotic phenotype and shortened long bones in adult mice. A proper longitudinal growth depends on a timely, well-coordinated vascularization and formation of the secondary ossification center (SOC) of the long bones epiphysis. Our results demonstrate that TRAP is not essential for the formation of the epiphyseal vascular network. Therefore, in wild type (Wt) controls as well as TRAP deficient (TRAP(-/-)) mutants vascularised cartilage canals are present from postnatal day (P) five. However, in the epiphysis of the TRAP(-/-) mice cartilage mineralization, formation of the marrow cavity and the SOC occur prematurely compared with the controls. In the mutant mice the entire growth plate is widened due to an expansion of the hypertrophic zone. This is not seen in younger animals but first detected at week (W) three and during further development. Moreover, an enhanced number of thickened trabeculae, indicative of the osteopetrotic phenotype, are observed in the metaphysis beginning with W three. Epiphyseal excavation was proposed as an important function of TRAP, and we examined whether TRAP deficiency affects this process. We therefore evaluated the marrow cavity volume (MCV) and the epiphyseal volume (EV) and computed the MCV to EV ratio (MCV/EV). We investigated developmental stages until W 12. Our results indicate that both epiphyseal excavation and establishment of the SOC are hardly impaired in the knockouts. Furthermore, no differences in the morphology of the epiphyseal bone trabeculae and remodeling of the articular cartilage layers are noted between Wt and TRAP(-/-) mice. We conclude that in long bones, TRAP is critical for the development of the growth plate and the metaphysis but apparently not for the epiphyseal vascularization, excavation, and establishment of the SOC.

[Effects of PTHrP and Notch signaling on the proliferation of epiphysis stem cells].

OBJECTIVE: To study the regulation of the proliferation of epiphysis stem cells by the PTHrP (parathyroid hormone related peptide) and Notch signaling systems. METHODS: An organ culture system of femurs of SD rat in 24 h after birth was employed. PTHrP (1 - 34) was used as the activator of the PTHrP signaling pathway and PTHrP (7 - 34) as the antagonist of PTH (parathyroid hormone)-receptor. For Notch signaling system, Jagged1/Fc was used as the activator and DAPT as its inhibitor. The femurs were cultured in DMEM (Dulbecco's modified Eagle's medium)/F12 medium while phosphate buffered saline was used for the control groups. Hematoxylin and eosin staining and bromodeoxyuridine analysis were used to analyze the length of the epiphysis stem cells zone and the proliferation of epiphysis stem cells. The expression of NICD (Notch intra-cellular domain) and Jagged1 were analyzed by immunohistochemistry. The epiphysis stem cells were transfected with the lentiviral vectors with rat PTHrP gene overexpression or inhibition properties, the cells transfected with the PGC-GFP-lentivirus or NC-GFP-lentivirus were used as control. Western blot was employed to detect the expression of NICD and Jagged1 genes. RESULTS: PTHrP (1 - 34) and Jagged1/Fc could dramatically elevate the rate of epiphysis stem cells zone by the whole growth plate length measurement while PTHrP (7 - 34) and DAPT could decrease the rate. Brdu analysis also showed that the number of proliferative epiphysis stem cells could be up-regulated by the PTHrP (1 - 34) or Jagged1/Fc signaling. By contrast, the treatment with PTHrP (7 - 34) or DAPT reduced the number of proliferative epiphysis stem cells. Immunohistochemistry and Western blot showed a significantly elevated expression of NICD and Jagged1 when PTHrP signaling was activated while a reductive expression of NICD and Jagged1 when PTHrP signaling was inactivated. CONCLUSION: Both of PTHrP and Notch signaling system could promote the proliferation of epiphysis stem cells. And the PTHrP signaling can stimulate Notch signaling to promote the proliferation of epiphysis stem cells.

Distribution of CD105 and CD166 positive cells in the proximal epiphysis of developing rat humerus.

The expression of cell surface receptors, CD105 and CD166, are characteristic of mesenchymal stem cells in cartilage. However, there is limited data regarding their immunolocalization in the cartilage of developing rat epiphysis. The purpose of this study was to determine the presence of CD105 and CD 166 positive cells in the proximal epiphysis of developing rat humerus and specify their zonal distribution with age. The tissues of rat humerus were taken on embryonic day 15 (E15), embryonic day 19 (E19), postnatal day 10 (PN10), postnatal day 20 (PN20) and adult rats and studied for the immunolocalization of CD105 and CD166. Our results showed that CD105 and CD166 positive cells were scattered in early stages of development of humerus epiphysis. For E15, only the hypertrophic zone was positive, whereas for E19 almost all zones of the epiphysis were positively stained for these markers. For PN10 and PN20, the CD105 and CD166 positive cells were mainly localized on the surface of the articular cartilage. In adult articular cartilage the CD105 and CD166 positive cells were localized in the superficial and transitional zones and in the upper regions of the deep zone. Our study provides evidence that in the developing cartilage tissue the localization of CD105 and CD166 positive cells is both dynamic and stage dependent, which may imply the existence of stem cell-like cells in cartilage from an early age to adult.

Size, neurochemistry, and segmental distribution of sensory neurons innervating the rat tibia.

Retrograde labeling has been used to identify sensory neurons in the lumbar dorsal root ganglia (DRG) that innervate the rat tibial periosteum, medullary cavity, and trabecular bone. The size, neurochemical profile [isolectin B4 (IB4) binding, substance P (SP), calcitonin gene-related peptide (CGRP), and NF200 immunoreactivity (-IR)], and segmental distribution of sensory neurons innervating each of these bony compartments are reported. After injections of fast blue into the periosteum, medullary cavity, and trabecular bone (epiphysis), retrogradely labeled neurons were observed throughout the ipsilateral (but not contralateral) lumbar DRG. They were predominantly small (<800 microm(2)) or medium-sized (800-1,800 microm(2)) neurons. CGRP-IR and SP-IR were found in 23% and 16% of the retrogradely labeled neurons, respectively. IB4 binding was observed in 20% and NF200-IR in 40% of the retrogradely labeled neurons. There were no significant differences in the percentage of neurons labeled with any one of the antisera following injections into each of the three bony compartments. To allow a direct comparison with sensory neurons innervating cutaneous tissues, injections of fast blue were also made into the skin overlying the tibia. The percentage of CGRP-IR neurons innervating bone was significantly lower than the percentage of CGRP-IR neurons innervating skin (ANOVA; P < 0.05). No other significant differences in the neurochemical profiles of neurons labeled from bone vs. skin were observed. The findings of the present study show that the periosteum, medullary cavity, and trabecular bone are all innervated by sensory neurons that have size and neurochemical profiles consistent with a role in nociception.

Incomplete digestion preserves chondrocytes from dedifferentiating in long-termed culture on plastic substrate.

Epiphyseal pieces from young rat's costal cartilage were predigested for 30min by hyaluronidase then digested by collagenase for 1h with gentle beating applied. Resulted grape-like chondrocytes connecting with the residual cartilage matrix were seeded in plastic culture dishes and 4 passages at about 12-days interval were carried out. Morphological observations were performed daily. Compared with completely isolated chondrocytes at the same passage, detection for collagen II, integrin-beta(1) and focal adhesion kinase by immunochemistry staining, Western Blot and RT-PCR were performed to evaluate the preservation of chondrocytic phenotype and cellular functions. Primary chondrocytes isolated by complete enzymatic digestion served as control. Completely isolated chondrocytes in the monolayer culture were ready to lose the chondrocytic phenotype marked by the down-regulation of collagen II secretion and specific morphological alterations which were characterized as the cells gradually became long and spindle-like from their originally rounded shape. In case of the incompletely digested chondrocytes, the expression of collagen II was stable during the whole experiment while extensive cell-cell contacts and matrix-cell connections were observed. Transcription and expression of integrin-beta(1) and FAK were active and paracrine of BMP-7 was positive. These results suggested stable chondrocytic phenotype. Conclusionly, by the incomplete digestion method, the requisite time for enzymatic isolation was reduced and chondrocytes with residual matrix were harvested instead of mono-cell suspension. Compared with the novel techniques, the incomplete digestion shortened the enzymatic procedure greatly and simplified the subculturing operations with less financial cost. Especially, as extracellular matrix was preserved, chondrocytes expressed stable phenotype in a rather long-termed culture.

Vitamin D and growth hormone regulate growth hormone/insulin-like growth factor (GH-IGF) axis gene expression in human fetal epiphyseal chondrocytes.

OBJECTIVE: Cell proliferation and gene expression regulation were studied in human fetal epiphyseal chondrocytes to ascertain the involvement of GH-IGF axis components in human fetal growth regulation by 1,25-dihydroxyvitamin D(3) (VitD) and growth hormone (GH). DESIGN: Chondrocytes from primary cultures were plated in serum-free medium for 48 h and incubated for a further 48 h with VitD (10(-11) to 10(-6)M) and/or IGF-I (100 ng/ml) and/or GH (500 ng/ml). We analyzed (3)H-thymidine incorporation into DNA and IGF-I, IGFBP-3, GHR, SOX9, COL2A1, aggrecan and COMP gene expression by real-time quantitative PCR. RESULTS: VitD dose-dependently and significantly inhibited (3)H-thymidine incorporation whereas GH had no effect on proliferation and, when combined with VitD, the same inhibition was observed as with VitD alone. IGF-I (100 ng/ml) significantly stimulated proliferation and opposed inhibition by VitD. VitD dose-dependently stimulated IGF-I (11.1+/-19.8 at VitD10(-6)M), IGFBP-3 (2.6+/-0.9), GHR (3.8+/-2.8) and COMP (1.5+/-0.6) expression whereas it inhibited SOX9 (0.7+/-0.2), COL2A1 (0.6+/-0.3) and aggrecan (0.6+/-0.2) expression and had no significant effect on IGF-II. IGF-I stimulated IGF-I, IGFBP-3, SOX9, COL2A1 and aggrecan expression and opposed COL2A1 and aggrecan gene expression inhibition by VitD. GH alone had no effect on gene expression whereas, in the presence of VitD, significantly-increased IGF-I expression stimulation was observed above values obtained with VitD alone (17.5+/-7.4). CONCLUSIONS: Our results suggest that VitD regulation of fetal growth cartilage could have consisted of parallel enhancing of cell differentiation and conditioning to a phenotype more sensitive to regulation by other hormones such as GH as shown by increased GHR and IGF-I expression, but not by IGF-II expression which was not regulated.

Transphyseal anterior cruciate ligament reconstruction using mesenchymal stem cells.

BACKGROUND: Conventional techniques for reconstruction of the anterior cruciate ligament in skeletally immature patients risk potential iatrogenic growth disturbance because of drilling across the physis. Animal models have demonstrated mixed results regarding growth disturbances from soft tissue grafts across the physis. HYPOTHESIS: Mesenchymal stem cells derived from bone marrow may be effective in preventing growth arrest after intra-articular anterior cruciate ligament reconstruction. STUDY DESIGN: Controlled laboratory study. MATERIAL AND METHODS: The anterior cruciate ligament was removed from 15 skeletally immature rabbits, which were divided into 3 groups: 5 rabbits (group 1) had only drilling of tunnels through the distal femoral and proximal tibial physes 5 (group 2) underwent drilling of the tunnels and reconstruction with an extensor digitorum communis autograft; and 5 (group 3) had drilling and reconstruction with an extensor digitorum communis autograft that had been seeded with mesenchymal stem cells. Radiographs were obtained every 3 weeks, and the animals were sacrificed 3 to 20 weeks after surgery. The surgically treated and contralateral control knees were salvaged, and each knee was examined grossly, radiographically, and histologically. RESULTS: A bone bridge spanned the physis in all nongrafted knees (group 1) by 3 weeks after surgery. In group 2, the extensor digitorum communis autograft seemed to slow but not prevent the development of bony bridges and angular deformities. In contrast, the mesenchymal stem cell-seeded grafts (group 3) appeared to provide a marked protective effect against growth arrest and angular deformity. CONCLUSION: The results of this study suggest that angular deformity and growth arrest that occur after drilling across the physis during anterior cruciate ligament reconstruction can be prevented or minimized by implanting mesenchymal stem cells onto the transphyseal soft tissue graft. CLINICAL RELEVANCE: These results may facilitate the development of strategies to prevent growth disturbances of the physis with intra-articular reconstructive procedures in pediatric patients.

[Immortalization of rat epiphysis cartilage cells induced by simian virus 40 large T antigen gene transfection].

OBJECTIVE: To establish immortalized epiphysis cartilage cell strains in order to provide a stable cell resource for cell substitution and gene therapies of growth retardation. METHODS: Plasmid pEGFP-IRES2-SV40LTag containing simian virus 40 large T antigen gene was transfected into primarily cultured epiphysis cartilage cells of the newborn rat using the lipofectin transfection method. Colonies were isolated by G418 selection and cultured to immortalized cell strains. Fibroblast growth factor receptor-3 (FGFR-3), anti-collagen type II and type X antibodies were used to identify cultured cells and to investigate the capability of differentiation of the transfected cells. SV40LTag expression in expanded cell strains was identified by RT-PCR, Southern blot and immunocytochemistry method. RESULTS: Anti-G418 cell clone was obtained, which was confirmed as FGFR-3 positive epiphysis cartilage cells with the capability of stable proliferation. mRNA and protein of SV40LTag were expressed in transfected cells after stable transfection. The transfected cells were expanded to immortalized cell strains and named as immortalized epiphysis cartilage cells. The immortalized cells were elliptic or triangular, with two or three short axons. The immortalized epiphysis cartilage cell strains had stable biological characters. CONCLUSIONS: SV40LTag gene transfection can immortalize epiphysis cartilage cells. The establishment of FGFR-3 positive immortalized epiphysis cartilage cell strains may provide a stable cell resource for cell substitution and gene therapies of growth retardation.