Translational approaches for bone construct

The contemporary mainstays for repair of craniofacial (CF) bony defects after trauma, osteonecrosis, and oncologic surgery are fixation, bone grafting and free tissue transfer. These work well in simple defects but are associated with morbidity and higher failure rates when used in complex reconstruction. Further, patients with medical comorbidities and elderly patients are often not eligible for these large procedures. Developing an osteogenic biosynthetic implant for CF reconstruction could alter standards of care in trauma management, osteonecrosis treatment, and oncologic surgical defects.FDA-approved rhBMP-2 is used to treat complex long bone fractures with good results and is frequently used in spinal fusion. Recent work, though, demonstrates this product provides little or no benefit compared with autologous tissue. Furthermore, rhBMP-2 has limited indications in CF reconstruction and is associated with poor outcomes when used off-label. While rhBMP-2 is the primary growth factor used clinically to support osteoinduction, PLGA scaffolds are the most common scaffolds used to support osteoconduction. However, concerns over inflammation secondary to PLGA’s acidic breakdown products have elucidated a need for natural polymer osteoconductive scaffolds. Our preliminary data shows that delivery of VEGF, BMP-6 and smoothened agonist (SAG) tethered to a novel polysaccharide scaffold (PS) significantly enhances bone regeneration in a critical-sized circular rat mandible defect in comparison with BMP-2 tethered to PS (PSBMP2). This PS scaffold developed in our laboratory not only overcomes the shortcomings of PLGA but also provides superior mechanical strength. We now want to investigate the bone regeneration potential of this novel treatment strategy using a clinically-relevant, critical-sized segmental rat mandibular defect model and compare its efficiency with Infuse bone graft (Medtronic), the only FDA-approved product available for CF bone regeneration.
Our central hypothesis is that treatment with PSVEGF+BMP6+SAG is superior to Infuse bone graft with regard to CF bone regeneration. To test this we will create 5 mm critical-sized segmental defects in rat mandibles and treat them with: rigid fixation only; fixation with Infuse bone graft; fixation with nude PS scaffold; fixation with PSSAG; fixation with PSBMP6+SAG; and fixation with PSBMP6+VEGF+SAG. We will assess bony regeneration using radiography, micro-CT, histology and mechanical strength testing.

Reconstruction of large mandibular defects in clinical contexts is challenging despite the characterization of pleiotropic morphogens that form bone. The need for alternatives to recombinant human bone morphogenetic proteins (hBMPs) is now felt more acutely following the reported complication, side-effects and performance failures associated with the clinical use of hBMP-2 and hBMP-7 (Baucus-Grassley 2012; Carragee et al. 2011). Contrary to all mammals tested so far, rectus abdominis implantation of recombinant human transforming growth factor-β3 (hTGF-β3) induces rapid and substantial bone formation in the Chacma baboon Papio ursinus. Bone forms via the BMPs pathway with hTGF-β3 controlling the induction of bone formation by regulating the expression of BMPs via Noggin expression. Expressed BMPs may escape Noggin’s antagonist action providing molecular insights into performance failure of hBMPs. Macroporous bioreactors pre-loaded with 250 µg hTGF-β3 showed the prominent induction of bone formation expanding within the rectus abdominis muscle. Full thickness mandibular defects 3 cm in length were created via an extra-oral approach in ten adult inbreed Papio ursinus after acclimatization at the Central Animal Services of the University. Defects were implanted with 250 µg hTGF-β3 per gr of human demineralized bone matrix. The study proposal is a long-term study to evaluate remodeling and maintenance of the regenerated mandibular defects with tissue harvest of mandibular blocks at 12 (four animals), 10 (two animals), 9 (two animals) and 7 (two animals) months for undecalcified histological analyses. Previous studies showed that plasma-sprayed crystalline titanium implants with a series of concavities initiated the induction of bone formation within the concavities. To study the effect of geometrically modified titanium surfaces without hydroxyapatite coating, two geometrically modified and two linear control implants are implanted in hTGF-β3 regenerates and at the edges of the mandibular osteotomies. Undecalcified mandibular blocks were harvested from the euthanized animals on days 15 and 30 after insertion of the titanium constructs in regenerated constructs 7, 9, 10 and 12 months post hTGF-β3 implantation. At tissue harvest, newly formed tissues around the neck of experimental and control implants were snap frozen for molecular analyses by qRT-PCR to study the early gene expression pathways controlling the induction of bone formation around geometrically modified and control titanium implants.

We are developing a liposomal formulation of recombinant human WNT3A protein that is intended to enhance the osteogenic properties of xenogeneic bone graft substitutes used in craniomaxillofacial reconstructive surgery.
Degenerative alveolar ridge changes are an inevitable part of aging, which also predictably occur following tooth loss, craniomaxillofacial trauma, periodontal disease, or tumor surgery. The resulting degenerative changes in skeletal structure and facial esthetics can significantly impact a patient’s quality of life. Alveolar ridge reconstruction procedures may be performed with autologous bone graft, bone substitutes, or combinations thereof but a range of early and late complications, coupled with extensive and rapid resorption result in a high failure rate 1,2. We are developing a pro-osteogenic human WNT3A protein therapeutic e.g., ART352-L for use in humans. We hypothesize that a combination of ART352-L with a xenograft material will result in robust, persistent vertical augmentation that will ultimately provide optimum esthetics and long-term implant stability.
To test this hypothesis, in Year 1 we will use a Wnt reporter strain of mice coupled with the development of a new alveolar ridge augmentation procedure to determine whether a combination of ART325-L and xenograft (Bio-Oss) results in significantly greater vertical volume of new bone compared to a xenograft alone. We have considerable experience with murine models of oral surgery including periosteal tunneling 3, xenografting 4, autografting 5-7, and implant osseointegration 8-14, therefore we anticipate being successful in developing this murine model. In Year 2, we will test whether the osteogenic effect elicited by ART352-L+xenograft material is on par or superior to the use of an autograft. In Year 3, we will demonstrate that the rate of bone resorption in the ART325-L+xenograft group is lower than the rate of bone resorption observed with the use of an autograft. If treatment with ART352-L enhances the osteogenic potential of xenograft material as expected, then the success of surgical procedures to increase vertical alveolar bone height would be increased, and their attendant complications would significantly decrease. Collectively, these pivotal studies will form the foundation for a clinical application of ART352-L to enhance vertical ridge reconstruction that has a direct benefit to an at-risk, elderly patient population.

PUBLICATION:
Chen J, Yuan x, Lib Z, Bahat DJ, Helms J.A.: Bioactivating a bone substitute accelerates graftincorporation in a murine model of vertical ridgeaugmentation, dental materials 36 (2020)1303–1313, doi: 10.1016/j.dental.2020.06.003