PTH Effects Bone Healing and Vasculogenesis in Calvaria Bone Allograft Model

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1 PTH Effects Bone Healing and Vasculogenesis in Calvaria Bone Allograft Model Dmitriy Sheyn, PhD 1, Doron Cohn-Yakubovich, BSc 2, Ilan Kallai, MSc 2, Susan Su, MD 1, Xiaoyu Da, MSc 1, Gadi Pelled, DMD, PhD 1,2, Wafa Tawackoli, PhD 1, Edward M. Schwarz, PhD 3, Dan Gazit, DMD, PhD 1,2, Zulma Gazit, PhD 1,2. 1 Cedars-Sinai Medical Center, Los Angeles, CA, USA, 2 Hebrew University of Jerusalem, Jerusalem, Israel, 3 University of Rochester, Rochester, NY, USA. Disclosures: D. Sheyn: None. D. Cohn-Yakubovich: None. I. Kallai: None. S. Su: None. X. Da: None. G. Pelled: None. W. Tawackoli: None. E.M. Schwarz: None. D. Gazit: None. Z. Gazit: None. Introduction There is a clear unmet medical need for the development of novel bone grafts for the treatment of craniofacial bone loss. Largescale bone defects in the cranial skeleton can result from congenital defects, acquired injuries, neurosurgical procedures, or infection. Unfortunately, successful spontaneous calvarial re-ossification rarely occurs, even in infants. Autologous bone grafts are not always available, and additional surgery must be performed for their harvest. Allografts are an attractive option for craniofacial bone reconstruction because of their high availability. Nevertheless, both experimental and clinical studies have shown that processed bone allografts fail to integrate with host bone due to formation of scar tissue. Recently, it was shown that daily teriparatide (recombinant human parathyroid hormone, PTH) treatment enhances integration of devitalized allograft in long bones and inhibits scar formation. We recently shown that PTH enhances allograft ossification in calvarial bone defect, in this study we are looking into the mechanism of this effect. We hypothesized that PTH treatment induces integration of allografts in cranial membranous bones via several mechanisms as depicted in Diagram 1: 1. Enhanced homing of MSCs to the site of injury and differentiation of MSC to osteoprogenitors; 2. Modulation of the vasculogenesis in the defect proximity; 3. Delay in mast cells infiltration thereby leading to more efficient bone formation. Diagram 1: Hypothesis Methods To pursue this hypothesis, we created circular calvarial bone defects in FVB/N mice. The mice were then divided into 2 groups and given implants of allografts, with or without daily PTH treatment (40 μg/kg/day). In vivo functional fluorescence imaging (FLI) of the blood vessels formation process was performed on day 7 post-implantation. The mice were imaged with FLI following injection of fluorescent probe IntegriSense (PerkinElmer Inc.), directed to bind integrin-αvβ3, a molecule that is highly

2 expressed in newly formed blood vessels. Bone formation and vasculogenesis were also analyzed using a micro-computed tomography scanner (μct 40; Scanco Medical AG, Brüttisellen, Switzerland), which was set at a nominal resolution of 12 µm. To further study the vascular tree structure in the graft-host junction we used intra-vital microscopy. Perfusion was evaluated using Laser Doppler imaging and scar tissue formation and the extent of fibrosis around the allograft were imaged to further validate the imaging data. Mouse bone marrow-derived MSCs were treated in vitro with PTH (100nM), RNA was isolated, and reverse transcription and qpcr was performed to evaluate the expression of the angiogenic gene angiopoietin 2 and the fibrogenic gene Ccn2/Ctgf. Statistical analysis was performed using a two-tailed homoscedastic Student t-test. Mast cells were detected by performing IF against two mast cell markers: mast cell protease 1 (MCP1, MAB5146, R&D Systems) and mast cell tryptase (MCT, LS-C18207, LifeSpan Biosciences, Inc., Seattle, WA); bonded primary antibodies were detected using secondary antibodies conjugated to AlexaFluor 488 (Invitrogen) or Cy3 (Jackson ImmunoResearch) with subsequent toluidine blue staining. Diagram 2. Experimental design Results and Discussion Our results show that the PTH therapy significantly increases bone formation in the allograft vicinity. Vasculogenesis detected by FLI a week after the surgery shows significant changes of the vascular tree formation following the PTH treatment (Fig. 1). Volumetric analysis of the blood vessels was performed using contrast agent injection and µct scanning revealed that large blood vessels were more likely to form in the untreated allografts, whereas in the PTH treated mice small blood vessel were more abundant (Fig. 2). Interestingly, when isolated bone marrow-derived MSCs, one of the main targets of the PTH therapy, were treated in vitro with PTH in osteogenic media, the expression of angiopoietin 2, responsible for large vessels formation, was downregulated on day 7 of treatment and Ccn2/Ctgf, a fibrogenic marker, was upregulated without the treatment (Fig 3). We also found that PTH modulated the infiltration of mast cells to the allograft proximity (Fig. 4). We strongly believe that mast cells play a fundamental role in fibrosis, scar tissue formation and vasculogenesis around the graft, consequently affecting the revitalization of the allograft.

3 Fig. 1. PTH modifies vasculogenesis in calvarial allograft, as shown by functional fluorescent imaging. FLI of IntegriSense integrin-αvβ3 -directed probe, a molecule that is highly expressed in newly formed blood vessels a week post surgery. Bars = SE, *p < 0.05, n = 3.

4 Fig. 2. PTH therapy affects the diameter of the newly formed blood vessel: with PTH treatment higher number of small vessels and lower number of large vessel were found using µct a week after surgery. Bars = SE, *p < 0.05, *** p < 0.001, n = 8.

5 Fig. 3. PTH downregulates angiogenic and fibrogenic gene expression of BM-MSCs in vitro. Angiopoietin-2, which responsible for large vessel formation, was downregulated on day 7 post surgery (A), whereas Ccn2/Ctgf, a well-established factor of scar tissue formation, was upregulated on day 10 of differentiation when PTH was absent in the osteogenic culture media. Bars = SE, *p < 0.05, n = 6.

6 Fig. 4. Mast cell infiltration is affected by PTH therapy, as detected by immunofluorescence (IF) and toluidine blue staining. Seven days after surgery, specimens were obtained from mice implanted with allografts and treated with PBS (placebo) (A & C) or PTH (B & D). After fixation and slicing, the sections were stained with antibodies against the mast cell markers mast cell protease 1 (MCP1) and mast cell tryptase (MCT) (A & B). Then the sections were stained with toluidine blue, which was useful for longitudinal mast cell quantification (C & D). Numbers of mast cells in both groups (E). Mast cells were manually counted in 36 toluidine blue-stained sections obtained from animals at 1, 2, and 3 weeks after surgery. Bars represent ± SE; * p < Conclusion In summary, PTH treatment enhances osteoprogenitor differentiation and augments bone formation around structural allografts. The precise mechanism is not clear, but we show that the formation of the vascular tree and the infiltration pattern of mast cells, associated with the formation of fibrotic tissue, are significantly modified following PTH therapy. We also show that PTH has direct effect on vasculogenesis via MSC recruitment. Results from this study and other ongoing experiments are designed to provide preclinical efficacy data to support a clinical trial of combined PTH-allograft therapy for bone repair in adult

7 patients suffering from calvaria trauma. Acknowledgements We acknowledge funding from the National Institutes of Health (NIDCR DEO19902 and the Administrative Supplement for DE019902). ORS 2014 Annual Meeting Poster No: 0052

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