Microtubule stabilization targeting regenerative chondrocyte cluster for cartilage regeneration.

Purpose: Chondrocytes (CHs) in cartilage undergo several detrimental events during the development of osteoarthritis (OA). However, the mechanism underlying CHs regeneration involved in pathogenesis is largely unknown. The aim of this study was to explore the underlying mechanism of regeneration of CHs involved in the pathological condition and the potential therapeutic strategies of cartilage repair. Methods and Materials: CHs were isolated from human cartilage in different OA stages and the high-resolution cellular architecture of human osteoarthritis was examined by applying single-cell RNA sequencing. The analysis of gene differential expression and gene set enrichment was utilized to reveal the relationship of cartilage regeneration and microtubule stabilization. Microtubule destabilizer (nocodazole) and microtubule stabilizer (docetaxel) treated-human primary CHs and rats cartilage defect model were used to investing the effects and downstream signaling pathway of microtubule stabilization on cartilage regeneration. Results: CHs subpopulations were identified on the basis of their gene markers and the data indicated an imbalance caused by an increase in the degeneration and disruption of CHs regeneration in OA samples. Interestingly, the CHs subpopulation namely CHI3L1+ CHs, was characterized by the cell regenerative capacity, stem cell potency and the activated microtubule (MT) process. Furthermore, the data indicated that MT stabilization was effective in promoting cartilage regeneration in rats with cartilage injury model by inhibiting YAP activity. Conclusion: These findings lead to a new understanding of CHs regeneration in the OA pathophysiology context and suggest that MT stabilization is a promising therapeutic target for OA and cartilage injury.

Compound 511 ameliorates MRSA-induced lung injury by attenuating morphine-induced immunosuppression in mice via PI3K/AKT/mTOR pathway.

BACKGROUND: Opioids are widely used in clinical practice. However, their long-term administration causes respiratory depression, addiction, tolerance, and severe immunosuppression. Traditional Chinese medicine (TCM) can alleviate opioid-induced adverse effects. Compound 511 is particularly developed for treating opioid addiction, based on Jiumi Liangfang, an ancient Chinese drug treatment and rehabilitation monograph completed in 1833 A.D. It is an herbal formula containing eight plants, each of them contributing to the overall pharmacological effect of the product: Panax ginseng C. A. Meyer (8.8%), Astragalus membranaceus (Fisch.) (18.2%), Datura metel Linn. (10.95%), Corydalis yanhusuo W. T. Wang (14.6%), Acanthopanar gracilistµlus W. W. Smith (10.95%), Ophiopogon japonicus (Linn. f.) Ker-Gawl. (10.95%), Gynostemma pentaphyllum (Thunb.) Makino (10.95%), Polygala arvensis Willd. (14.6%). This formula effectively ameliorates opioid-induced immunosuppression. However, the underlying mechanism remains unclear. PURPOSE: To reveal the effects of Compound 511 on the immune response of morphine-induced immunosuppressive mice and their potential underlying molecular mechanism. This study provides information for a better clinical approach and scientific use of opioids. METHODS: Immunosuppression was induced in mice by repeated morphine administration. Th1/Th2/Th17/Treg cell levels were measured using flow cytometry. Splenic transcription factors of Th1/Th2/Th17/Treg and outputs of the regulatory PI3K/AKT/mTOR signaling pathway were determined. Subsequently, methicillin-resistant Staphylococcus aureus (MRSA) was administered intranasally to morphine-induced immunosuppressive mice pretreated with Compound 511. Their lung inflammatory status was assessed using micro-computer tomography (CT), hematoxylin and eosin (H&E) staining, and enzyme-linked immunosorbent assay (ELISA). RESULTS: Compared to morphine, Compound 511 significantly decreased the immune organ indexes of mice, corrected the Th1/Th2 and Treg/Th17 imbalance in the immune organs and peripheral blood, reduced the mRNA levels of FOXP3 and GATA3, and increased those of STAT3 and T-bet in the spleen. It improved immune function and reduced MRSA-induced lung inflammation. CONCLUSION: Compound 511 ameliorates opioid-induced immunosuppression by regulating the balance of Th1/Th2 and Th17/Treg via PI3K/AKT/mTOR signaling pathway. Thus, it effectively reduces susceptibility of morphine-induced immunosuppressive mice to MRSA infection.

Molecular crosstalk between articular cartilage, meniscus, synovium, and subchondral bone in osteoarthritis.

AIMS: Osteoarthritis (OA) is a common degenerative joint disease worldwide, which is characterized by articular cartilage lesions. With more understanding of the disease, OA is considered to be a disorder of the whole joint. However, molecular communication within and between tissues during the disease process is still unclear. In this study, we used transcriptome data to reveal crosstalk between different tissues in OA. METHODS: We used four groups of transcription profiles acquired from the Gene Expression Omnibus database, including articular cartilage, meniscus, synovium, and subchondral bone, to screen differentially expressed genes during OA. Potential crosstalk between tissues was depicted by ligand-receptor pairs. RESULTS: During OA, there were 626, 97, 1,060, and 2,330 differentially expressed genes in articular cartilage, meniscus, synovium, and subchondral bone, respectively. Gene Ontology enrichment revealed that these genes were enriched in extracellular matrix and structure organization, ossification, neutrophil degranulation, and activation at different degrees. Through ligand-receptor pairing and proteome of OA synovial fluid, we predicted ligand-receptor interactions and constructed a crosstalk atlas of the whole joint. Several interactions were reproduced by transwell experiment in chondrocytes and synovial cells, including TNC-NT5E, TNC-SDC4, FN1-ITGA5, and FN1-NT5E. After lipopolysaccharide (LPS) or interleukin (IL)-1ß stimulation, the ligand expression of chondrocytes and synovial cells was upregulated, and corresponding receptors of co-culture cells were also upregulated. CONCLUSION: Each tissue displayed a different expression pattern in transcriptome, demonstrating their specific roles in OA. We highlighted tissue molecular crosstalk through ligand-receptor pairs in OA pathophysiology, and generated a crosstalk atlas. Strategies to interfere with these candidate ligands and receptors may help to discover molecular targets for future OA therapy.Cite this article: Bone Joint Res 2022;11(12):862-872.

Articular fibrocartilage-targeted therapy by microtubule stabilization.

The fibrocartilage presented on the joint surface was caused by cartilage injury or degeneration. There is still a lack of effective strategies for fibrocartilage. Here, we hypothesized that the fibrocartilage could be viewed as a raw material for the renewal of hyaline cartilage and proposed a previously unidentified strategy of cartilage regeneration, namely, "fibrocartilage hyalinization." Cytoskeleton remodeling plays a vital role in modifying the cellular phenotype. We identified that microtubule stabilization by docetaxel repressed cartilage fibrosis and increased the hyaline cartilage extracellular matrix. We further designed a fibrocartilage-targeted negatively charged thermosensitive hydrogel for the sustained delivery of docetaxel, which promoted fibrocartilage hyalinization in the cartilage defect model. Moreover, the mechanism of fibrocartilage hyalinization by microtubule stabilization was verified as the inhibition of Sparc (secreted protein acidic and rich in cysteine). Together, our study suggested that articular fibrocartilage-targeted therapy in situ was a promising strategy for hyaline cartilage repair.

Nonlinear Energy Evolution Characteristics of Diorite Examined by Triaxial Loading-Unloading and Acoustic Emission Tests.

Acoustic emission (AE) is often accompanied by the propagation of internal microcracks in loaded rock samples, and it essentially reflects microinstability phenomena driven by energy redistribution under stress. In this paper, loading and unloading tests were carried out to investigate the internal nonlinear damage evolution characteristics of diorite samples under different unloading confining-pressure rates. The nonlinear mechanical characteristics of the strain energy sequence of diorite were studied by applying nonlinear dynamics and basic chaos theory and MATLAB software. Moreover, the evolution characteristics of AE counts and AE energy of rock samples were investigated, and their microcrack-propagation modes were analyzed based on the RA−AF scatter distribution of AE and a two-dimensional Gaussian mixture model. Finally, according to the evolution characteristics of energy and AE, the nonlinear damage evolution mechanism of diorite under loading and unloading conditions was revealed. The results show that, before the loading and unloading peak strength, when the strain-energy-promotion coefficient, r, is equal to 1 or changes in the ranges of 1−3, 3−3.57, and ≥3.57, the strain-energy evolution of diorite presents the characteristics of supercritical stability, nonlinear stability, period-doubling stability, and chaos, respectively. Meanwhile, the greater the rate of the unloading confining pressure, the earlier the period-doubling bifurcation and chaotic mechanical behavior will occur. After loading and unloading peak strength, the sudden decrease of high-density AE counts and AE energy or the sudden transition of the strain-energy-promotion coefficient from >0 to <0 can be used as an important criterion for the complete failure of rock samples.

Single cell RNA-seq analysis identifies ferroptotic chondrocyte cluster and reveals TRPV1 as an anti-ferroptotic target in osteoarthritis.

BACKGROUND: Osteoarthritis (OA) is the most common degenerative joint disease primarily characterized by cartilage destruction. The aim of this study was to investigate the role, molecular characteristics and potential therapeutic target of chondrocyte ferroptosis in the pathogenesis of OA. METHODS: The expression of ferroptotic hallmarks (iron and lipid peroxidation accumulation, glutathione deletion) were analyzed in paired intact and damaged cartilages from OA patients. Single cell RNA sequencing (scRNA-seq) analysis was performed on 17,638 chondrocytes to verify the presence, investigate the molecular signatures and unveil the potential therapeutic target of ferroptotic chondrocyte cluster in human OA cartilages. Destabilization of medial meniscus (DMM)-induced OA model and tert-butyl hydroperoxide (TBHP)-treated primary mouse chondrocytes and human cartilage explants were used to evaluate the protective effect of pharmacologically activated transient receptor potential vanilloid 1 (TRPV1). The downstream molecular mechanisms of TRPV1 was further investigated in glutathione peroxidase 4 (Gpx4) heterozygous genetic deletion mice (Gpx4+/-). FINDINGS: The concentrations of iron and lipid peroxidation and the expression of ferroptotic drivers in the damaged areas of human OA cartilages were significantly higher than those in the intact cartilage. scRNA-seq analysis revealed a chondrocyte cluster characterized by preferentially expressed ferroptotic hallmarks and genes, namely ferroptotic chondrocyte cluster. Comprehensive gene set variation analysis revealed TRPV1 as an anti-ferroptotic target in human OA cartilage. Pharmacological activation of TRPV1 significantly abrogated cartilage degeneration by protecting chondrocytes from ferroptosis. Mechanistically, TRPV1 promoted the expression of GPX4, and its anti-ferroptotic role was largely mitigated in the OA model of Gpx4+/- mice. INTERPRETATION: TRPV1 activation protects chondrocytes from ferroptosis and ameliorates OA progression by upregulating GPX4. FUNDING: National Key R&D Program of China (2018YFC1105904), Key Program of NSFC (81730067), National Science Foundation of China (81772335, 81941009, 81802196), Natural Science Foundation of Jiangsu Province, China (BK20180127), Jiangsu Provincial Key Medical Talent Foundation, Six Talent Peaks Project of Jiangsu Province (WSW-079).

Microtubule Stabilization Enhances the Chondrogenesis of Synovial Mesenchymal Stem Cells.

Mesenchymal stem cells (MSCs) are well known for their multi-directional differentiation potential and are widely applied in cartilage and bone disease. Synovial mesenchymal stem cells (SMSCs) exhibit a high proliferation rate, low immunogenicity, and greater chondrogenic differentiation potential. Microtubule (MT) plays a key role in various cellular processes. Perturbation of MT stability and their associated proteins is an underlying cause for diseases. Little is known about the role of MT stabilization in the differentiation and homeostasis of SMSCs. In this study, we demonstrated that MT stabilization via docetaxel treatment had a significant effect on enhancing the chondrogenic differentiation of SMSCs. MT stabilization inhibited the expression of Yes-associated proteins (YAP) and the formation of primary cilia in SMSCs to drive chondrogenesis. This finding suggested that MT stabilization might be a promising therapeutic target of cartilage regeneration.

Nitric Oxide Nanomotor Driving Exosomes-Loaded Microneedles for Achilles Tendinopathy Healing.

The microneedle (MN) provides a promising strategy for transdermal delivery of exosomes (EXO), in which the therapeutic effects and clinical applications are greatly reduced by the fact that EXO can only partially reach the injury site by passive diffusion. Here, we designed a detachable MN array to deliver EXO modified by a nitric oxide nanomotor (EXO/MBA) for Achilles tendinopathy (AT) healing. With the releasing of EXO/MBA, l-arginine was converted to nitric oxide by NOS or ROS as the driving force. Benefiting from the motion ability and the property of MPC tending to lower pH, EXO could accumulate at the injury site more efficiently. This work demonstrated that EXO/MBA-loaded MN notably suppressed the inflammation of AT, facilitated the proliferation of tendon cells, increased the expression of Col1a, and prevented extracellular matrix degradation, indicating its potential value in enthesiopathy healing and other related biomedical fields.

Circular RNA expression profiling in the nucleus accumbens: Effects of electroacupuncture treatment on morphine-induced conditioned place preference.

Electroacupuncture (EA) has been developed on the basis of traditional Chinese acupuncture. EA can suppress craving in opioid addicts and opioid-seeking responses in rodents. However, the molecular mechanism of EA on the rewarding properties of morphine and craving responses is not known. Here, we have applied a conditioned place preference paradigm in mice to measure morphine-induced rewarding effects along with EA treatment. Circular RNAs (circRNAs) can function as micro RNA (miRNA) sponges to effectively regulate gene expression levels. CircRNA profiling within the nucleus accumbens (NAc) was performed in EA-treated and sham-treated mice. Following RNAseq, data were analyzed by gene ontology (GO) and Kyoto Encyclopedia Genes and Genomes (KEGG) tools. We identified 112 significantly differentially expressed circRNAs, including 51 that were up-regulated and 61 that were down-regulated. Our bioinformatics analyses show that these differentially expressed circRNAs map into pathways that are mainly involved with renin secretion and the cGMP-PKG signaling. We further constructed a circRNA-miRNA network that predicts the potential roles of the differentially expressed circRNAs and the interaction of circRNAs with miRNAs. Our secondary sequencing and bioinformatics analysis in the NAc after EA treatment on morphine-induced CPP provides putative novel targets on molecular mechanisms involved in morphine reinforcement and possibly craving.

Analgesic Effect of Zanthoxylum nitidum Extract in Inflammatory Pain Models Through Targeting of ERK and NF-κB Signaling.

BACKGROUND: Zanthoxylum nitidum (Roxb.) DC., also named Liang Mianzhen (LMZ), one kind of Chinese herb characterized with anti-inflammatory and relieving pain potency, which is widely used to treat injuries, rheumatism, arthralgia, stomach pain and so on in China. But its mechanism related to the anti-hyperalgesic has not been reported. The aim of this study was to investigate the analgesic activity of Liang Mianzhen on mice with Complete Freund adjuvant (CFA)-induced chronic inflammatory pain. Meanwhile, the peripheral and central mechanisms of analgesic effect of Liang Mianzhen were further examined via observing the effects of Liang Mianzhen on the signal pathway associated with inflammatory induced hyperalgesia. METHODS: The inflammatory pain model was established by intraplantar injection of CFA in C57BL/6J mice. After 1 day of CFA injection, the mice were treated with LMZ (100 mg/kg) for seven consecutive days, and the behavioral tests were measured after the daily intragastric administration of LMZ. The morphological changes on inflamed paw sections were determined by hematoxylin eosin (HE) staining. Changes in the mRNA expression levels of tumor necrosis factor (TNF-α), interleukin-6 (IL-6), interleukin-1ß (IL-1ß) and nuclear factor κB p65 (NF-κBp65) were measured on day seven after CFA injection by using real-time quantitative PCR analysis and enzyme linked immunosorbent assay (ELISA) method, respectively. Moreover, immunohistochemistry and western blotting were used to detect extracellular regulated protein kinases 1/2 (ERK1/2) and NF-κB signal pathway activation. RESULTS: The extract of LMZ (100 mg/kg) showed a significant anti-inflammatory and analgesic effect in the mice model. The paw edema volume was significantly reduced after the administration of LMZ compared to CFA group, as well as the paw tissues inflammatory damage was relived and the numbers of neutrophils in mice was reduced significantly. The CFA-induced mechanical threshold and thermal hyperalgesia value were significant improved with LMZ treatment at day three to day seven. We also found the mRNA levels of TNF-α, IL-1ß, IL-6 and NF-κBp65 were down-regulate after 7 days from the LMZ treatment compared to CFA group. Meanwhile, LMZ significantly suppressed over-expression of the phosphorylation of ERK1/2 and NF-κBp65 in peripheral and central. CONCLUSION: The present study suggests that the extract of LMZ attenuates CFA-induced inflammatory pain by suppressing the ERK1/2 and NF-κB signaling pathway at both peripheral and central level.

Kuntai capsule attenuates premature ovarian failure through the PI3K/AKT/mTOR pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Kuntai capsule (KTC), a type of herb formulas, was first described in the book of Shang Han Za Bing Lun in the third century. KTC has been widely used for the clinical treatment of menopausal syndrome. Considering that premature ovarian failure is also known as premature menopause, this study was designed to investigate the effects and mechanisms of KTC on a mouse model of premature ovarian failure. MATERIALS AND METHODS: Forty-five female C57BL/6 mice were chosen for this study. Fifteen of the mice were separated into the Control group. The remaining thirty were used to establish the premature ovarian failure model by injecting intraperitoneally with 75 mg/kg cyclophosphamide and then by randomly dividing the mice into two groups. One group was considered the Model group, the other group treated with the Kuntai capsule intragastrically every day for one week called the KTC group. After treatment, mice were sacrificed for sampling. The ovaries morphology of mice was observed by hematoxylin and eosin (HE) staining, and all follicles were counted under microscope. Western blotting was used to detect the PI3K/AKT/mTOR pathway activation. Serum follicle-stimulating hormone (FSH), luteinizing hormone (LH), estradiol (E2) and anti-mullerian hormone (AMH)levels were measured by enzyme-linked immunosorbent assay (ELISA). The fertility was observed by giving treated mice 8 weeks for breeding. RESULTS: We found that primordial follicle counts were increased in the KTC group compared to the Model group. The phosphorylation of PI3K, AKT, mTOR, 4E-BP1 and S6K in the KTC group significantly reduced compared to Model group. Serum FSH and LH levels in the KTC group were decreased compared to the Model group, while, serum E2 and AMH levels in the KTC group were increased compared with the Model group. The litter size in the KTC group was improved compared to Model group. CONCLUSIONS: The KTC showed protective potentials of ovarian reserve and fertility to attenuate premature ovarian failure, which was relatively associated with activation of the PI3K/AKT/mTOR signaling pathway.

Electro-acupuncture attenuates the mice premature ovarian failure via mediating PI3K/AKT/mTOR pathway.

AIMS: Electro-acupuncture (EA) is frequently recommended as a complementary therapy for premature ovarian failure (POF) in the clinical. However, little information exists about its potential treatment mechanisms. The study was designed to observe the effect of EA to ovarian function and fertility in POF mice model, and investigated its potential mechanisms on PI3K/AKT/mTOR signaling pathway. MATERIALS AND METHODS: Forty-five female C57/BL6 mice were divided into the Control, the Model and the EA group. The ovaries morphology of mice was observed by hematoxylin and eosin (HE) staining, and all follicles were counted under microscope. The protein expression of PI3K, phospho-PI3K, AKT, phospho-AKT, mTOR, phospho-mTOR, S6, phospho-S6, 4E-BP1 and phospho-4E-BP1 were detected by western blotting. The data was presented as the ratio of phosphorylation protein to total protein. Serum follicle-stimulating hormone (FSH), luteinizing hormone (LH), estradiol (E2) and anti-Mullerian hormone (AMH) levels were measured by enzyme-linked immunosorbent assay (ELISA). The fertility was observed by giving treated mice 8 weeks for breeding. KEY FINDINGS: We found that primordial follicle counts were increased in EA group compared to Model group. The phosphorylation of PI3K, AKT, mTOR, 4E-BP1 and S6K in EA group significantly reduced compared to Model group. Serum FSH and LH levels in EA group were decreased compared to Model group, while, serum E2 and AMH levels in EA group were increased compared with Model group. The litter size in EA group was improved compared to Model group. SIGNIFICANCE: The effects of EA on the PI3K/AKT/mTOR signaling pathway may represent one of the mechanisms involved in attenuating the mice POF.

Sex Associated Differential Expressions of the Alternatively Spliced Variants mRNA of OPRM1 in Brain Regions of C57BL/6 Mouse.

BACKGROUND/AIMS: Opiates are potent analgesics but their clinical use is limited by sex-associated side effects, such as drug tolerance, opioid-induced hyperalgesia and withdrawal reaction. OPRM1, as the main receptor of opioids, plays an important role in the pharmacological process of opioids in rodents and human. We have previously investigated OPRM1, the µ opioid receptor gene, which have dozens of alternatively spliced variants probably correlating with opioid-induced effects in brain regions of four inbred mouse strains and demonstrated the strain-specific expressions of these splice variants. Also, within a strain, the regional expression patterns of some of the variants were similar while others were opposite. Thus, we are aiming to seek out the relationship between sex differences and these alternatively spliced variants. METHODS: The present studies follow a SYBR green quantitative PCR (qPCR) which we had used before to examine the expression of OPRM1 splice variant mRNAs in selected brain regions of male and female C57BL/6 mice. Sex-associated differences in baseline latency, opioid-induced tolerance, analgesia and addiction were examined and determined by Tail-flick test, jumps and statistical analysis. RESULTS: The mRNA levels of opioid receptor gene splice variants in male and female mice showed significant differences among the brain regions, implying region-specific alternative splicing of the OPRM1 gene, which was consistent with our previous study. More importantly, the complete mRNA expression profiles of the OPRM1 splice variants was also gender-specific, suggesting a sexual influence on OPRM1 alternative splicing. CONCLUSION: In brief, we put forward that the distinctions among baseline latency, opioid-induced tolerance, analgesia and physical dependence in male and female mice might correlate with sex associated differential expressions of OPRM1 gene.

Exogenous PTHrP Repairs the Damaged Fracture Healing of PTHrP+/- Mice and Accelerates Fracture Healing of Wild Mice.

Bone fracture healing is a complicated physiological regenerative process initiated in response to injury and is similar to bone development. To demonstrate whether an exogenous supply of parathyroid hormone-related protein (PTHrP) helps in bone fracture healing, closed mid-diaphyseal femur fractures were created and stabilized with intramedullary pins in eight-week-old wild-type (WT) PTHrP+/+ and PTHrP+/- mice. After administering PTHrP for two weeks, callus tissue properties were analyzed at one, two, and four weeks post-fracture (PF) by various methods. Bone formation-related genes and protein expression levels were evaluated by real-time reverse transcriptase-polymerase chain reaction and Western blots. At two weeks PF, mineral density of callus, bony callus areas, mRNA levels of alkaline phosphatase (ALP), type I collagen, Runt-related transcription factor 2 (Runx-2), and protein levels of Runx-2 and insulin-like growth factor-1 decreased in PTHrP+/- mice compared with WT mice. At four weeks PF, total collagen-positive bony callus areas, osteoblast number, ALP-positive areas, and type I collagen-positive areas all decreased in PTHrP+/- mice. At both two and four weeks PF, tartrate-resistant acid phosphatase-positive osteoclast number and surface decreased a little in PTHrP+/- mice. The study indicates that exogenous PTHrP provided by subcutaneous injection could redress impaired bone fracture healing, leading to mutation of activated PTHrP by influencing callus areas, endochondral bone formation, osteoblastic bone formation, and bone turnover.

Exogenous Parathyroid Hormone-Related Peptide Promotes Fracture Healing in Lepr(-/-) Mice.

Diabetic osteoporosis continues to surge worldwide, increasing the risk of fracture. We have previously demonstrated that haploinsufficiency of endogenous parathyroid hormone-related peptide (PTHrP) impairs fracture healing. However, whether an exogenous supply of PTHrP can repair bone damage and accelerate fracture healing remains unclear. This study aimed to assess the efficacy and safety of PTHrP in healing fractures. Standardized mid-diaphyseal femur fractures were generated in 12-week-old wild-type and leptin receptor null Lepr(-/-) mice. After administration of PTHrP for 2 weeks, callus tissue properties were analyzed by radiography, micro-computed tomography, histology, histochemistry, immunohistochemistry, and molecular biology techniques. At 2 weeks post-fracture, cartilaginous callus areas were reduced, while total callus and bony callus areas were increased in PTHrP-treated Lepr(-/-) animals and control wild-type mice, compared with vehicle-treated Lepr(-/-) mice. The following parameters were enhanced both in Lepr(-/-) mice after treatment with PTHrP and vehicle-treated wild-type animals, compared with vehicle-treated Lepr(-/-) mice: osteoblast numbers; tissue alkaline phosphatase (ALP) and Type I collagen immunopositive areas; mRNA levels of ALP, Type I collagen, osteoprotegerin, and receptor activator for nuclear factor-κ B ligand; protein levels of Runt-related transcription factor 2 and insulin-like growth factor-1; and the number and surface of osteoclasts. In conclusion, exogenous PTHrP by subcutaneous injection promotes fracture repair in Lepr(-/-) mice by increasing callus formation and accelerating cell transformation: upregulated osteoblastic gene and protein expression, increased endochondral bone formation, osteoblastic bone formation, and osteoclastic bone resorption. However, complete repair was not obtained in PTHrP-treated Lepr(-/-) mice as in control wild-type animals.