Growth and repair factors, osteoactivin, matrix metalloproteinase and heat shock protein 72, increase with resolution of inflammation in musculotendinous tissues in a rat model of repetitive grasping

Authors

Nagat Frara, Samir M. Abdelmagid, Michael Tytell, Mamta Amin, Steven N. Popoff, Fayez F. Safadi and Mary F. Barbe

Abstract

Expression of the growth factor osteoactivin (OA) increases during tissue degeneration and regeneration, fracture repair and after denervation-induced disuse atrophy, concomitant with increased matrix metalloproteinases (MMPs). However, OA’s expression with repetitive overuse injuries is unknown. The aim of this study was to evaluate: 1) OA expression in an operant rat model of repetitive overuse; 2) expression of MMPs; 3) inflammatory cytokines indicative of injury or inflammation; and 4) the inducible form of heat shock protein 70 (HSPA1A/HSP72) as the latter is known to increase during metabolic stress and to be involved in cellular repair. Young adult female rats performed a high repetition negligible force (HRNF) food retrieval task for up to 6 weeks and were compared to control rats.

Flexor digitorum muscles and tendons were collected from 22 young adult female rats performing a HRNF reaching task for 3 to 6 weeks, and 12 food restricted control (FRC) rats. OA mRNA levels were assessed by quantitative polymerase chain reaction (qPCR). OA, MMP-1, -2, -3, and -13 and HSP72 protein expression was assayed using Western blotting. Immunohistochemistry and image analysis was used to evaluate OA and HSP72 expression. ELISA was performed for HSP72 and inflammatory cytokines.

Flexor digitorum muscles and tendons from 6-week HRNF rats showed increased OA mRNA and protein expression compared to FRC rats. MMP-1, -2 and -3 progressively increased in muscles whereas MMP-1 and -3 increased in tendons with HRNF task performance. HSP72 increased in 6-week HRNF muscles and tendons, compared to controls, and co-localized with OA in the myofiber sarcolemma. IL-1alpha and beta increased transiently in tendons or muscles in HRNF week 3 before resolving in week 6.

The simultaneous increases of OA with factors involved in tissue repair (MMPs and HSP72) supports a role of OA in tissue regeneration after repetitive overuse.

Link to Article

http://dx.doi.org/10.1186/s12891-016-0892-3

Enamel Matrix Derivative Promotes Healing of a Surgical Wound in the Rat Oral Mucosa

Authors

Tal Maymon-Gil, Evgeny Weinberg, Carlos Nemcovsky, Miron Weinreb

Abstract

Background: Enamel Matrix Proteins (EMPs) play a role in enamel formation and the development of the periodontium. Sporadic clinical observations of periodontal regeneration treatments with Enamel Matrix Derivative (EMD), a commercial formulation of EMPs, suggest it also promotes post-surgical healing of soft tissues. In vitro studies showed that EMD stimulates various cellular effects, which could potentially enhance wound healing. This study examined the in-vivo effects of EMD on healing of an oral mucosa surgical wound in rats.

Methods: A bilateral oral mucosa wound was created via a crestal incision at the anterior edentulous maxilla of Sprague-Dawley rats. Full thickness flaps were raised and following suturing, EMD was injected underneath the soft tissues on one side while the EMD vehicle was injected in the contralateral side. Animals were sacrificed after 5 or 9 days and the wound area was subjected to histological and immunohistochemical analysis of epithelial gap, number of macrophages, blood vessels, proliferating cells and collagen content in the connective tissue. Gene expression analysis was also conducted 2 days after surgery.

Results: EMD had no effect on epithelial gap of the wound. On both days 5 and 9 EMD treatment increased significantly the number of blood vessels and the collagen content. EMD also enhanced (by 20-40%) the expression of transforming growth factor (TGF) β1 and TGFβ2, vascular endothelial growth factor (vEGF), interleukin-1β (IL-1β), matrix metalloproteinase-1 (MMP-1), Versican and Fibronectin.

Conclusions: EMD improves oral mucosa incisional wound healing by promoting formation of blood vessels and collagen fibers in the connective tissue.

Link to Article

http://dx.doi.org/10.1902/jop.2016.150567

Systemic Administration of Sclerostin Antibody Enhances Bone Morphogenetic Protein-Induced Femoral Defect Repair in a Rat Model

Authors

Brian A. Tinsley, MD; Alex Dukas, MD; Michael J. Pensak, MD; Douglas J. Adams, PhD; Amy H. Tang, BS; Michael S. Ominsky, PhD; Hua Zhu Ke, PhD; Jay R. Lieberman, MD

Abstract

Background: Recombinant human bone morphogenetic protein (rhBMP)-2 is a potent osteoinductive agent; however, its clinical use has been reduced because of safety and efficacy concerns. In preclinical studies involving a critical-sized defect in a rat model, sclerostin antibody (Scl-Ab) treatment increased bone formation within the defect but did not result in reliable healing. The purpose of the current study was to evaluate bone repair of a critical-sized femoral defect in a rat model with use of local implantation of rhBMP-2 combined with systemic administration of Scl-Ab.

Methods: A critical-sized femoral defect was created in rats randomized into three treatment groups: local rhBMP-2 and systemic Scl-Ab (Scl + BMP), local rhBMP-2 alone, and collagen sponge alone (operative control). The Scl + BMP group received local rhBMP-2 (10 μg) on a collagen sponge placed within the defect intraoperatively and then twice weekly injections of Scl-Ab (25 mg/kg) administered postoperatively. The femora were evaluated at twelve weeks with use of radiography, microcomputed tomography (microCT), histomorphometric analysis, and biomechanical testing.

Results: At twelve weeks, all Scl + BMP and rhBMP-2 only samples were healed. No femora healed in the operative control group. Histomorphometric analysis demonstrated more bone in the Scl + BMP samples than in the samples treated with rhBMP-2 alone (p = 0.029) and the control samples (p = 0.003). MicroCT revealed that the Scl + BMP group had a 90% greater bone volume within the defect region compared with the rhBMP-2 group and a 350% greater bone volume compared with the operative control group (p < 0.001). Biomechanical testing showed that the group treated with Scl + BMP had greater torsional strength and rigidity compared with the rhBMP-2 group (p < 0.001 and p = 0.047) and the intact femoral control group (p < 0.001). Torque to failure was lower in the rhBMP-2 group compared with the intact femoral control group (p < 0.002). Mean energy to failure was higher in the Scl + BMP samples compared with the rhBMP-2 only samples (p = 0.001).

Conclusions: In a critical-sized femoral defect in a rat model, local rhBMP-2 combined with systemic administration of Scl-Ab resulted in more robust healing that was stronger and more rigid than results for rhBMP-2 alone and intact nonoperative femora.

Clinical Relevance: Our study demonstrated that combining an osteoinductive agent with a systemically administered antibody that promotes bone formation can enhance bone repair and has potential as a therapeutic regimen in humans.

Link to Article

http://dx.doi.org/10.2106/JBJS.O.00171

IGF-1 Regulates Vertebral Bone Aging Through Sex-Specific and Time-Dependent Mechanisms

Authors

Nicole M Ashpole PhD, Jacquelyn C Herron MS, Matthew C Mitschelen, Julie A Farley, Sreemathi Logan PhD, Han Yan PhD, Zoltan Ungvari MD/PhD, Erik L. Hodges, Anna Csiszar MD/PhD, Yuji Ikeno MD/PhD, Mary Beth Humphrey PhD and William E Sonntag PhD

Abstract

Advanced aging is associated with increased risk of bone fracture, especially within the vertebrae, which exhibit significant reductions in trabecular bone structure. Aging is also associated with a reduction in circulating levels of insulin-like growth factor (IGF-1). Studies have suggested that the reduction in IGF-1 compromises healthspan, while others report that loss of IGF-1 is beneficial as it increases healthspan and lifespan. To date, the effect of decreases in circulating IGF-1 on vertebral bone aging has not been thoroughly investigated. Here, we delineate the consequences of a loss of circulating IGF-1 on vertebral bone aging in male and female Igff/f mice. IGF-1 was reduced at multiple specific time points during the mouse lifespan- early in postnatal development (crossing albumin-Cre mice with Igff/f mice), or early adulthood, and late adulthood using hepatic-specific viral vectors (AAV8-TBG-Cre). Vertebrae bone structure was analyzed at 27 months of age using microCT and quantitative bone histomorphometry. Consistent with previous studies, both male and female mice exhibited age-related reductions in vertebral bone structure. In male mice, reduction of circulating IGF-1 induced at any age did not diminish vertebral bone loss. Interestingly, early-life loss of IGF-1 in females resulted in a 67% increase in vertebral bone volume fraction, as well as increased connectivity density and increased trabecular number. The maintenance of bone structure in the early-life IGF-1-deficient females was associated with increased osteoblast surface and an increased ratio of osteoprotegerin/receptor-activator of NFkB-ligand levels in circulation. Within 3 months of a loss of IGF-1, there was a 2.2 fold increase in insulin receptor expression within the vertebral bones of our female mice, suggesting that local signaling may compensate for the loss of circulating IGF-1. Together, these data suggest the age-related loss of vertebral bone density in females can be reduced by modifying circulating IGF-1 levels early in life.

Link to Article

http://dx.doi.org/10.1002/jbmr.2689

RAP-011 augments callus formation in closed fractures in rats

Authors

Alyson Morse, Tegan Cheng, Lauren Peacock, Kathy Mikulec, David Little, and Aaron Schindeler

Abstract

ACE-011 is a bone anabolic agent generated by fusing the extracellular domain of the Activin Type 2A receptor (ActRIIA) to an IgG-Fc. The orthopedic utility of ACE-011 was investigated using a murine analogue, RAP-011. Initially, a rat closed fracture model was tested using bi-weekly (biw) 10 mg/kg RAP-011. RAP-011 significantly increased callus length and callus bone volume (BV, +43% at 6w, p < 0.01). The polar moment of inertia was calculated to be substantively increased (+80%, p < 0.01), however mechanical bending tests showed a more modest increase in maximum load to failure (+24%, p < 0.05). Histology indicated enhanced appositional bone growth, but it was hypothesized that reduced remodeling, evidenced by decreased serum CTX (-16% at 6w, p < 0.01), could be compromising bone quality in the callus. A second closed fracture study was performed to examine lower ‘pulse’ [RAP-011(p)] and ‘sustained’ [RAP-011(s)] regimens of biw 0.6mg/kg × 2, 0.35mg/kg × 3 and 0.18mg/kg × 2, 0.1mg/kg × 7 respectively, compared with PTH(1-34) (25µg/kg/d) and vehicle controls. RAP-011 treatments gave modest increases in callus length and callus BV at 6w (p < 0.01), but did not achieve an increase in maximum load over vehicle. In summary, RAP-011 is effective in promoting bone formation during repair, but optimizing callus bone quality will require further investigation.

Link To Article

http://dx.doi.org/10.1002/jor.22985

Cortical Bone Histomorphometry in Male Femoral Neck: The Investigation of Age-Association and Regional Differences

Authors

Xiaoyu Tong, Inari S. Burton, Hanna Isaksson, Jukka S. Jurvelin, Heikki Kröger

Abstract

Low bone volume and changes in bone quality or microarchitecture may predispose individuals to fragility fractures. As the dominant component of the human skeleton, cortical bone plays a key role in protecting bones from fracture. However, histological investigations of the underlying structural changes, which might predispose to fracture, have been largely limited to the cancellous bone. The aim of this study was to investigate the age-association and regional differences of histomorphometric properties in the femoral neck cortical bone. Undecalcified histological sections of the femoral neck (n = 20, aged 18–82 years, males) were cut (15 μm) and stained using modified Masson-Goldner stain. Complete femoral neck images were scanned, and cortical bone boundaries were defined using our previously established method. Cortical bone histomorphometry was performed with low (×50) and high magnification (×100). Most parameters related to cortical width (Mean Ct.Wi, Inferior Ct.Wi, Superior Ct.Wi) were negatively associated with age both before and after adjustment for height. The inferior cortex was the thickest (P < 0.001) and the superior cortex was the thinnest (P < 0.008) of all cortical regions. Both osteonal size and pores area were negatively associated with age. Osteonal area and number were higher in the antero-inferior area (P < 0.002) and infero-posterior area (P = 0.002) compared to the postero-superior area. The Haversian canal area was higher in the infero-posterior area compared to the postero-superior area (P = 0.002). Moreover, porosity was higher in the antero-superior area (P < 0.002), supero-anterior area (P < 0.002) and supero-posterior area (P < 0.002) compared to the infero-anterior area. Eroded endocortical perimeter (E.Pm/Ec.Pm) correlated positively with superior cortical width. This study describes the changes in cortical bone during ageing in healthy males. Further studies are needed to investigate whether these changes explain the increased susceptibility to femoral neck fractures.

Link To Article

http://dx.doi.org/10.1007/s00223-015-9957-9