Magnetic resonance and diffusion tensor imaging of the adolescent rabbit growth plate of the knee.

PURPOSE: To assess the ability of MRI-DTI to evaluate growth plate morphology and activity compared with that of histomorphometry and micro-CT in rabbits. METHODS: The hind limbs of female rabbits aged 16, 20, and 24 wk (n = 4 per age group) were studied using a 9.4T MRI scanner with a multi-gradient echo 3D sequence and DTI in 14 directions (b-value = 984 s/mm2 ). After MRI, the right and left hind limb were processed for histological analysis and micro-CT, respectively. The Wilcoxon signed-rank test was used to evaluate the height and volume of the growth plate. Intraclass correlation and Pearson correlation coefficient were used to evaluate the association between DTI metrics and age. RESULTS: The growth plate height and volume were similar for all modalities at each time point and age. Age was correlated with all tractography and DTI metrics in both the femur and tibia. A correlation was also observed between all the metrics at both sites. Tract number and volume declined with age; however, tract length did not show any changes. The fractional anisotropy color map showed lateral diffusion centrally in the growth plate and perpendicular diffusion in the hypertrophic zone, as verified by histology and micro-CT. CONCLUSION: MRI-DTI may be useful for evaluating the growth plates.

The synovial microenvironment suppresses chondrocyte hypertrophy and promotes articular chondrocyte differentiation.

During the development of the appendicular skeleton, the cartilaginous templates undergo hypertrophic differentiation and remodels into bone, except for the cartilage most adjacent to joint cavities where hypertrophic differentiation and endochondral bone formation are prevented, and chondrocytes instead form articular cartilage. The mechanisms that prevent hypertrophic differentiation and endochondral bone formation of the articular cartilage have not been elucidated. To explore the role of the synovial microenvironment in chondrocyte differentiation, osteochondral allografts consisting of articular cartilage, epiphyseal bone, and growth plate cartilage from distal femoral epiphyses of inbred Lewis rats expressing enhanced green fluorescent protein from a ubiquitous promoter were transplanted either in inverted or original (control) orientation to matching sites in wildtype littermates, thereby allowing for tracing of transplanted cells and their progenies. We found that no hypertrophic differentiation occurred in the growth plate cartilage ectopically placed at the joint surface. Instead, the transplanted growth plate cartilage, with time, remodeled into articular cartilage. This finding suggests that the microenvironment at the articular surface inhibits hypertrophic differentiation and supports articular cartilage formation. To explore this hypothesis, rat chondrocyte pellets were cultured with and without synoviocyte-conditioned media. Consistent with the hypothesis, hypertrophic differentiation was inhibited and expression of the articular surface marker lubricin (Prg4) was dramatically induced when chondrocyte pellets were exposed to synovium- or synoviocyte-conditioned media, but not to chondrocyte- or osteoblast-conditioned media. Taken together, we present evidence for a novel mechanism by which synoviocytes, through the secretion of a factor or factors, act directly on chondrocytes to inhibit hypertrophic differentiation and endochondral bone formation and promote articular cartilage formation. This mechanism may have important implications for articular cartilage development, maintenance, and regeneration.

Optimized protocols for in situ hybridization, immunohistochemistry, and immunofluorescence on skeletal tissue.

Assessment of gene and protein expression in tissue sections is instrumental in medical research. However, this is often challenging to perform on skeletal tissues that require prolonged decalcification and have poor adhesion to slides. In this study, we optimized selected steps of in situ hybridization (ISH), immunohistochemistry (IHC), and immunofluorescence (IF) for formalin fixed and decalcified skeletal tissues. Sections from distal femur of 6-, 8- and 14- week-old rats injected with BrdU with or without a hemizygous eGFP transgene expressed under the control of a ubiquitous promotor were used. We report that proteinase K digestion is critical for the sensitivity of ISH, as concentrations that were too strong and too mild both resulted in loss of signal. In addition, intensified RNase A digestion removed nonspecific riboprobe-mRNA hybrids. Furthermore, enzymatic antigen retrieval using proteinase K provided more consistent results in IHC and can therefore be a useful alternative to heat induced epitope retrieval (HIER) for skeletal tissues where such treatment often damages the morphology. A mild proteinase K digestion also improved IF detection of GFP and worked well for double labeling IF of GFP and osteocalcin on frozen sections of formalin fixed and decalcified rat bones while maintaining morphology. In summary, this study provides strategies to improve protocols for enzymatic digestion in ISH, IHC, and IF for skeletal tissues and also demonstrates the importance of careful optimization and validation with the use of these techniques.

Rat perichondrium transplanted to articular cartilage defects forms articular-like, hyaline cartilage.

OBJECTIVE: Perichondrium autotransplants have been used to reconstruct articular surfaces destroyed by infection or trauma. However, the role of the transplanted perichondrium in the healing of resurfaced joints has not been investigated. DESIGN: Perichondrial and periosteal tissues were harvested from rats hemizygous for a ubiquitously expressed enhanced green fluorescent protein (EGFP) transgene and transplanted into full-thickness articular cartilage defects at the trochlear groove of distal femur in wild-type littermates. As an additional control, cartilage defects were left without a transplant (no transplant control). Distal femurs were collected 3, 14, 56, 112 days after surgery. RESULTS: Tracing of transplanted cells showed that both perichondrium and periosteum transplant-derived cells made up the large majority of the cells in the regenerated joint surfaces. Perichondrium transplants contained SOX9 positive cells and with time differentiated into a hyaline cartilage that expanded and filled out the defects with Col2a1-positive and Col1a1-negative chondrocytes and a matrix rich in proteoglycans. At later timepoints the cartilaginous perichondrium transplants were actively remodeled into bone at the transplant-bone interface and at post-surgery day 112 EGFP-positive perichondrium cells at the articular surface were positive for Prg4. Periosteum transplants initially lacked SOX9 expression and despite a transient increase in SOX9 expression and chondrogenic differentiation, remained Col1a1 positive, and were continuously thinning as periosteum-derived cells were incorporated into the subchondral compartment. CONCLUSIONS: Perichondrium and periosteum transplanted to articular cartilage defects did not just stimulate regeneration but were themselves transformed into cartilaginous articular surfaces. Perichondrium transplants developed into an articular-like, hyaline cartilage, whereas periosteum transplants appeared to produce a less resilient fibro-cartilage.

Identification of Novel Cytotoxic T Lymphocyte Epitopes of Drug- Resistance Related Protein InhA from Mycobacterium tuberculosis.

BACKGROUND: Tuberculosis (TB) caused by Mycobacterium tuberculosis (MTB), especially the drug-resistant MTB, poses serious challenges to human healthcare worldwide. Cytotoxic T lymphocytes (CTLs) play a vital role in immune defense against MTB. OBJECTIVE: To identify novel CTL epitopes that could induce cellular immunity against MTB infections. METHODS: The HLA-A*0201 restricted CTL epitopes of the drug-resistant protein InhA from MTB were predicted by online algorisms and synthesized by the Fmoc solid phase method. The candidate peptides were used to induce CTLs from human peripheral blood mononuclear cells (PBMCs) of HLA-A*0201 healthy donors and the HLA-2.1/Kb mice. IFN-γ productions of CTLs were detected by enzyme linked immunospot assay (ELISPOT), flow cytometry and enzyme-linked immunosorbent assay (ELISA), and cytotoxicity was analyzed by lactate dehydrogenase (LDH) assay. RESULTS: A group of 4 epitopes were screened out with high affinities to HLA-A*0201. ELISPOT and flow cytometry analysis indicated these peptides significantly induced that IFN-γ release of CTLs from the HLA-A*0201+/PPD+ donors, as the mutant analogues had more potent stimulation effects. LDH assay showed that CTLs from PPD+ donors and the immunized mice exhibited significant cytotoxicity and low cross-reactivity. ELISA analysis revealed comparative levels of IFN-γ were released by CTLs isolated from the mice spleen. CONCLUSION: Our study has identified 4 novel CTL epitopes of InhA that could elicit potent CTL immunity, establishing a foundation for the development of multivalent peptide vaccines against the drug-resistant MTB.