Botulinum Toxin Induces Muscle Paralysis and Inhibits Bone Regeneration in Zebrafish

Intramuscular administration of Botulinum toxin (BTx) has been associated with impaired osteogenesis in diverse conditions of bone formation (eg, development, growth, and healing), yet the mechanisms of neuromuscular‐bone crosstalk underlying these deficits have yet to be identified. Motivated by the emerging utility of zebrafish (Danio rerio) as a rapid, genetically tractable, and optically transparent model for human pathologies (as well as the potential to interrogate neuromuscular‐mediated bone disorders in a simple model that bridges in vitro and more complex in vivo model systems), in this study, we developed a model of BTx‐induced muscle paralysis in adult zebrafish, and we examined its effects on intramembranous ossification during tail fin regeneration. BTx administration induced rapid muscle paralysis in adult zebrafish in a manner that was dose‐dependent, transient, and focal, mirroring the paralytic phenotype observed in animal and human studies. During fin regeneration, BTx impaired continued bone ray outgrowth, morphology, and patterning, indicating defects in early osteogenesis. Further, BTx significantly decreased mineralizing activity and crystalline mineral accumulation, suggesting delayed late‐stage osteoblast differentiation and/or altered secondary bone apposition. Bone ray transection proximal to the amputation site focally inhibited bone outgrowth in the affected ray, implicating intra‐ and/or inter‐ray nerves in this process. Taken together, these studies demonstrate the potential to interrogate pathological features of BTx‐induced osteoanabolic dysfunction in the regenerating zebrafish fin, define the technological toolbox for detecting bone growth and mineralization deficits in this process, and suggest that pathways mediating neuromuscular regulation of osteogenesis may be conserved beyond established mammalian models of bone anabolic disorders. © 2014 American Society for Bone and Mineral Research.     

Heterotopic Bone Formation Following Resurfacing Total Hip Arthroplasty

Heterotopic bone (HO), a rare association with total hip arthroplasty (THA), has recently been shown to be more of a problem with resurfacing hip arthroplasty (RHA). It has been speculated to be the result of greater soft tissue dissection required for this procedure. HO most commonly develops in males and patients with bilateral disease. To better understand if this problem does occur in RHA, groups of patients with RHA on one side and conventional THA on the other were evaluated. We retrospectively identified 45 patients that had RHA on one side and conventional cemented THA on the other. Follow-up has been up to 25 years. HO was graded at every clinical visit using the Brooker Classification. In the RHA group, there were 32 hips without evidence of HO, ten with grade 1, and three with grades 2 or 3. In the THA group, there were 36 hips without any HO, eight with grade 1, and one with grade 2. There was no statistical difference between either types of hip arthroplasty in HO formation overall or in the development of more severe grades. Motion was not significantly affected with the more severe grades. Our data obtained from a bilateral patient model suggests that RHA does not predispose to a greater development of HO. In doing a RHA, one should be aware that there might be an increased likelihood of HO in males with bilateral disease.     

Celecoxib and Heterotopic Bone Formation After Total Hip Arthroplasty

We assessed the effectiveness of celecoxib in the prevention of heterotopic ossification (HO) following primary total hip replacement (THR). We studied 170 consecutive THRs. Sixty-three patients received celecoxib after surgery (200 mg twice/daily) for 28 days and 84 did not. HO was more common in non-celecoxib patients than in the celecoxib-group at 3, 6, and 12 months (P = 0.005, 0.004 and 0.01, respectively). At 1 year, fewer celecoxib recipients had Brooker classes II or III. None of the celecoxib patients developed HO Brooker class IV, while 2% in the non-celecoxib group did. No patient discontinued treatment or had revision for aseptic loosening. A short course of celecoxib for pain aids in the prevention of HO after primary THR, and could be a useful and safe option that does not interfere with anticoagulation.     

Muscle Changes Can Account for Bone Loss After Botulinum Toxin Injection

Studies to date have assumed that botulinum toxin type A (BTX) affects bone indirectly, through its action on muscle. We hypothesized that BTX has no discernable effect on bone morphometry, independent of its effect on muscle. Therefore, we investigated whether BTX had an additional effect on bone when combined with tenotomy compared to tenotomy in isolation. Female BALB/c mice (n = 73) underwent one of the following procedures in the left leg: BTX injection and Achilles tenotomy (BTX-TEN), BTX injection and sham surgery (BTX-sham), Achilles tenotomy (TEN), or sham surgery (sham). BTX groups were injected with 20 μL of BTX (1 U/100 g) in the posterior lower hindlimb. At 4 weeks, muscle cross-sectional area (MCSA) and tibial bone morphometry were assessed using micro-CT. Each treatment, other than sham, resulted in significant muscle and bone loss (P < 0.05). BTX-TEN experienced the greatest muscle loss (23–45% lower than other groups) and bone loss (20–30% lower bone volume fraction than other groups). BTX-sham had significantly lower MCSA and bone volume fraction than TEN and sham. After adjusting for differences in MCSA, there were no significant between-group differences in bone properties. We found that BTX injection resulted in more adverse muscle and bone effects than tenotomy and that effects were amplified when the procedures were combined. However, between-group differences in bone could be accounted for by MCSA. We conclude that any independent effect of BTX on bone morphometry is likely small or negligible compared with the effect on muscle.     

Factors Associated with Heterotopic Bone Formation in Cemented Total Hip Prostheses

A series of 237 total hip replacements were analysed by means of a computer program. High body weight and postoperative fever, as well as several indications of a technically demanding operation, occurred significantly more often in the group of patients which developed paraarticular ossification postoperatively. the results point to a causal relation between tissue trauma and heterotopic bone formation.

We wish to underline the importance of a gentle handling of tissues in the performance of a total hip replacement if the rate of ectopic bone is to be reduced to a minimum.     

Platelet-Rich Plasma Application and Heterotopic Bone Formation Following Total Hip Arthroplasty

ABSTRACT

Activated blood platelets play a critical, early role in the wound healing response by releasing several types of growth factors at the site of injury which mediate the initial stages of tissue repair. Autologously derived platelet-rich plasma has been applied during surgery as a healing aid and some studies have shown benefit with total joint arthroplasty procedures such as in the knee. However, little has been published regarding the use of platelet-rich plasma during total hip arthroplasty. The hip is especially prone to develop islands of heterotopic bone following arthroplasty which can lead to pain, limited motion, and even ankylosis of the joint. If this condition is exacerbated by platelet-rich plasma, this could present a barrier to the use of this adjuvant in total hip arthroplasty. This retrospective, controlled clinical study examined the effect of platelet rich plasma application during closure following total hip arthroplasty on heterotopic ossification. By one year, 21.3% of the control patients developed heterotopic bone (91 patients, 94 hips, Brooker grades I–III) compared to 12.9% of the treatment patients (76 patients, 85 hips, Brooker grades I–II). These differences were not significant (p = 0.478, power = 0.90). Thus, the use of platelet-rich plasma in this procedure does not appear to influence the incidence or severity of heterotopic ossification which should help to justify further clinical research to more fully understand whether this autologous blood product has a role in total hip arthroplasty.     

Acidosis Inhibits Bone Formation by Osteoblasts In Vitro by Preventing Mineralization

The negative effect of acidosis on the skeleton has been known for almost a century. Bone mineral serves an important pathophysiologic role as a reserve of hydroxyl ions to buffer systemic protons if the kidneys and lungs are unable to maintain acid-base balance within narrow physiologic limits. Extracellular hydrogen ions are now thought to be the primary activation signal for osteoclastic bone resorption, and osteoclasts are very sensitive to small changes in pH within the pathophysiologic range. Herein, we investigated the effects of acidosis on osteoblast function by using mineralized bone nodule-forming primary osteoblast cultures. Osteoblasts harvested from neonatal rat calvariae were cultured up to 21 days in serum-containing medium, with ascorbate, beta-glycerophosphate and dexamethasone. pH was manipulated by addition of 5 to 30 mmol/L HCl and monitored by blood gas analyzer. Abundant, matrix-containing mineralized nodules formed in osteoblast cultures at pH 7.4, but acidification progressively reduced mineralization of bone nodules, with complete abolition at pH 6.9. Osteoblast proliferation and collagen synthesis, assessed by 3H-thymidine and 3H-proline incorporation, respectively, were unaffected by pH in the range 7.4 to 6.9; no effect of acidification on collagen ultrastructure and organization was evident. The apoptosis rate of osteoblasts, assessed by the enrichment of nucleosomes in cell lysates, was also unaffected by pH within this range. However, osteoblast alkaline phosphatase activity, which peaked strongly near pH 7.4, was reduced eight-fold at pH 6.9. Reducing pH to 6.9 also downregulated messenger ribonucleic acid (mRNA) for alkaline phosphatase, but upregulated mRNA for matrix Gla protein, an inhibitor of mineralization. The same pH reduction is associated with two-and four-fold increases in Ca2+ and PO4(3-) solubility for hydroxyapatite, respectively. Our results show that acidosis exerts a selective, inhibitory action on matrix mineralization that is reciprocal with the osteoclast activation response. Thus, in uncorrected acidosis, the deposition of alkaline mineral in bone by osteoblasts is reduced, and osteoclast resorptive activity is increased in order to maximize the availability of hydroxyl ions in solution to buffer protons.     

Combined Effects of Botulinum Toxin Injection and Hind Limb Unloading on Bone and Muscle

Bone receives mechanical stimulation from two primary sources, muscle contractions and external gravitational loading; but the relative contribution of each source to skeletal health is not fully understood. Understanding the most effective loading for maintaining bone health has important clinical implications for prescribing physical activity for the treatment or prevention of osteoporosis. Therefore, we investigated the relative effects of muscle paralysis and reduced gravitational loading on changes in muscle mass, bone mineral density, and microarchitecture. Adult female C57Bl/6J mice (n = 10/group) underwent one of the following: unilateral botulinum toxin (BTX) injection of the hind limb, hind limb unloading (HLU), both unilateral BTX injection and HLU, or no intervention. BTX and HLU each led to significant muscle and bone loss. The effect of BTX was diminished when combined with HLU, though generally the leg that received the combined intervention (HLU+BTX) had the most detrimental changes in bone and muscle. We found an indirect effect of BTX affecting the uninjected (contralateral) leg that led to significant decreases in bone mineral density and deficits in muscle mass and bone architecture relative to the untreated controls; the magnitude of this indirect BTX effect was comparable to the direct effect of BTX treatment and HLU. Thus, while it was difficult to definitively conclude whether muscle force or external gravitational loading contributes more to bone maintenance, it appears that BTX-induced muscle paralysis is more detrimental to muscle and bone than HLU.     

Heterotopic Bone Formation About the Hip Undergoes Endochondral Ossification: A Rabbit Model

Background

Heterotopic ossification (HO) occurs most commonly after trauma and surgery about the hip and may compromise subsequent function. Currently available animal models describing the cellular progression of HO are based on exogenous osteogenic induction agents and may not reflect the processes following trauma.

Questions/purposes

We therefore sought to characterize the histologic progression of heterotopic bone formation in an animal model that recapitulates the human condition without the addition of exogenous osteogenic material.

Methods

We used a rabbit model that included intramedullary instrumentation of the upper femur and ischemic crush injury of the gluteal muscle. Bilateral surgical induction procedures were performed on 30 animals with the intention of inciting the process of HO; no supplemental osteogenic stimulants were used. Three animals were sacrificed at each of 10 predetermined times between 1 day and 26 weeks postoperatively and the progression of tissue maturation was graded histologically using a five-item scale.

Results

Heterotopic bone reliably formed de novo and consistently followed a pathway of endochondral ossification. Chondroid elements were found in juxtaposition with immature woven bone in all sections that contained mature osseous elements.

Conclusions

These results establish that HO occurs in an animal model mimicking the human condition following surgical trauma about the hip; it is predictable in its histologic progression and follows a pathway of endochondral bone formation.

Clinical Relevance

By showing a consistent pathway of endochondral ossification leading to ectopic bone formation, this study provides a basis for understanding the mechanisms by which HO might be mitigated by interventions.     

Rosiglitazone Inhibits Bone Regeneration and Causes Significant Accumulation of Fat at Sites of New Bone Formation

Thiazolidinediones (TZDs), peroxisome proliferator-activated receptor gamma activators, and insulin sensitizers represent drugs used to treat hyperglycemia in diabetic patients. Type 2 diabetes mellitus (T2DM) is associated with a twofold increase in fracture risk, and TZDs use increases this risk by an additional twofold. In this study, we analyzed the effect of systemic administration of the TZD rosiglitazone on new bone formation in two in vivo models of bone repair, a model of drilled bone defect regeneration (BDR) and distraction osteogenesis (DO) and a model of extended bone formation. Rosiglitazone significantly inhibited new endosteal bone formation in both models. This effect was correlated with a significant accumulation of fat cells, specifically at sites of bone regeneration. The diminished bone regeneration in the DO model in rosiglitazone-treated animals was associated with a significant decrease in cell proliferation measured by the number of cells expressing proliferating cell nuclear antigen and neovascularization measured by both the number of vascular sinusoids and the number of cells producing proangiogenic vascular endothelial growth factor at the DO site. In summary, rosiglitazone decreased new bone formation in both BDR and DO models of bone repair by mechanisms which include both intrinsic changes in mesenchymal stem cell proliferation and differentiation and changes in the local environment supporting angiogenesis and new bone formation. These studies suggest that bone regeneration may be significantly compromised in T2DM patients on TZD therapy.     

CRMP4 Inhibits Bone Formation by Negatively Regulating BMP and RhoA Signaling

We identified the neuroprotein collapsing response mediator protein‐4 (CRMP4) as a noncanonical osteogenic factor that regulates the differentiation of mouse bone marrow skeletal stem cells (bone marrow stromal stem cells [mBMSCs]) into osteoblastic cells. CRMP4 is the only member of the CRMP1–CRMP5 family to be expressed by mBMSCs and in osteoprogenitors of both adult mouse and human bones. In vitro gain‐of‐function and loss‐of‐function of CRMP4 in murine stromal cells revealed its inhibitory effect on osteoblast differentiation. In addition, Crmp4‐deficient mice (Crmp4^–/–) displayed a 40% increase in bone mass, increased mineral apposition rate, and bone formation rate, compared to wild‐type controls. Increased bone mass in Crmp4^–/– mice was associated with enhanced BMP2 signaling and BMP2‐induced osteoblast differentiation in Crmp4^–/– osteoblasts (OBs). Furthermore, Crmp4^–/– OBs exhibited enhanced activation of RhoA/focal adhesion kinase (FAK) signaling that led to cytoskeletal changes with increased cell spreading. In addition, Crmp4^–/– OBs exhibited increased cell proliferation that was mediated via inhibiting cyclin‐dependent kinase inhibitor 1B, p27^Kip1 and upregulating cyclin D1 expression which are targets of RhoA signaling pathway. Our findings identify CRMP4 as a novel negative regulator of osteoblast differentiation. © 2016 American Society for Bone and Mineral Research.     

Ciliary Neurotrophic Factor Inhibits Bone Formation and Plays a Sex-Specific Role in Bone Growth and Remodeling

Ciliary neurotrophic factor (CNTF) receptor (CNTFR) expression has been described in osteoblast-like cells, suggesting a role for CNTF in bone metabolism. When bound to CNTF, neuropoietin (NP), or cardiotrophin-like-cytokine (CLC), CNTFR forms a signaling complex with gp130 and the leukemia inhibitory factor receptor, which both play critical roles in bone cell biology. This study aimed to determine the role of CNTFR-signaling cytokines in bone. Immunohistochemistry detected CNTF in osteoblasts, osteocytes, osteoclasts, and proliferating chondrocytes. CNTFR mRNA was detected in primary calvarial osteoblasts and was upregulated during osteoblast differentiation. Treatment of osteoblasts with CNTF or CLC, but not NP, significantly inhibited mineralization and osterix mRNA levels. Twelve-week-old male CNTF ^−/− mice demonstrated reduced femoral length, cortical thickness, and periosteal circumference; but femoral trabecular bone mineral density (Tb.BMD) and tibial trabecular bone volume (BV/TV) were not significantly different from wild-type, indicating a unique role for CNTF in bone growth in male mice. In contrast, female CNTF ^−/− femora were of normal width, but femoral Tb.BMD, tibial BV/TV, trabecular number, and trabecular thickness were all increased. Female CNTF ^−/− tibiae also demonstrated high osteoblast number and mineral apposition rate compared to wild-type littermates, and this was intrinsic to the osteoblast lineage. CNTF is expressed locally in bone and plays a unique role in female mice as an inhibitor of trabecular bone formation and in male mice as a stimulus of cortical growth.     

肌替代术治疗股四头肌瘫痪

目的 观察采用肌替代术治疗股四头肌瘫痪的效果。方法 60例因脊髓灰质炎后遗股四头肌瘫痪患,采用不同术式的肌肉替代转移重建股四头肌功能。结果 经追踪随访,有效56例（占93％）,无效4例（占7％）。结论 股二头肌－半腱肌代股四头肌术效果最好。对下肢广泛而严重肌瘫痪的病例,在无肌肉可替代的情况下,人工碳纤维替代术也是一种可行的方法。     

Outcomes of Chemical Component Paralysis Using Botulinum Toxin for Incisional Hernia Repairs

Background

Botulinum toxin A (BTX) confers flaccid paralysis and pain modulation when injected into a muscle. We hypothesized that long-term paralysis of the abdominal wall musculature (i.e., chemical component paralysis, CCP) would benefit incisional hernia repair (IHR) by decreasing postoperative pain, the use of opioid analgesia, and thus opioid-related side effects.

Methods

Adult patients who underwent elective IHR with preoperative CCP (n = 22) were compared to concurrent matched controls (n = 66, 1:3 ratio) based on age (±5 years), sex, body mass index (±5 kg/m²), history of hernia recurrence (0 vs. ≥1), and type of repair (open vs. laparoscopic). BTX was injected under ultrasonographic guidance into the transversus abdominis and internal and external oblique muscles at three sites bilaterally (300 units total).

Results

Patients who underwent IHR with CCP used significantly less opioid analgesia (mean ± SD morphine equivalents) when compared to controls on hospital days (HDs) 2 and 5: HD2, 48 ± 27 versus 87 ± 41; HD5, 17 ± 16 versus 48 ± 45. Likewise, CCP patients reported significantly less pain (visual analogue scale 1–10) when compared to controls on HD2 (5.2 ± 1.5 vs. 6.8 ± 2) and HD4 (3.6 ± 1.2 vs. 5.2 ± 1.9): all p < 0.007 (Bonferroni adjusted). There was no difference in postoperative complications (surgical site, 9 vs. 14 %), opioid-related adverse events (ileus 5 vs. 5 %), hospital stay (4 ± 3 vs. 3 ± 2 days), or hernia recurrence (18 months mean follow-up: 9 vs. 9 %).

Conclusions

Despite similar multimodality treatment of postoperative pain after IHR, patients who underwent CCP required significantly less opioid analgesia and reported less pain.     

Histomorphometric Analysis of Heterotopic Bone Formed by Stromal-Osteogenic Subpopulations

MBA-15.4 and MBA-15.6 cell lines are marrow stromal clonal subpopulations and represent various stages of differentiation of the osteoblastic family. These cells vary in terms of morphology, proliferation rate, synthesis of matrix proteins as collagen and noncollagenous proteins, and by their responses to hormones and growth factors. Their differential properties directly reflect the clonal cells' ability to form bone in vivo. When the cells were transplanted at an ectopic site, under the kidney capsule, MBA-15.4 line formed small foci of bone whereas MBA-15.6 cell line formed massive woven bone during the same period of time. In this study, we focused on the histomorphometric analysis of ectopic ossicles formed by the clonal cell lines. Assessments of bone mass changes involved measurements of cellular components, osteoid, and formation of primary bone. The bony tissue formed was condensed, no hemopoiesis was noted, and the ossicle was not remodeled. The histology studies were used for quantitative analysis of the ossicle formation and describe the dynamics of ossicles formed by the individual cell types. Received: 9 August 1995 / Accepted: 12 April 1996