A study of the influence of genetic variance and sex on the density and thickness of the calvarial bone in collaborative cross mice

Background:Bone microarchitecture is affected by multiple genes,each having a small effect on the external appearance.It is thus challenging to characterize the genes and their specific effect on bone thickness and porosity.The purpose of this study was to assess the heritability and the genetic variation effect,as well as the sex effect on the calvarial bone thickness(Ca.Th)and calvarial porosity(%PoV)using the Collaborative Cross(CC)mouse population.Methods:In the study we examined the parietal bones of 56 mice from 9 lines of CC mice.Morphometric parameters were evaluated using microcomputed tomography(μCT)and included Ca.Th and%PoV.We then evaluated heritability,genetic versus environmental variance and the sex effect for these parameters.Results:Our morphometric analysis showed that Ca.Th and%PoV are both significantly different among the CC lines with a broad sense heritability of 0.78 and 0.90,respectively.The sex effect within the lines was significant in line IL111 and showed higher values of Ca.Th and%PoV in females compared to males.In line IL19 there was a borderline sex effect in Ca.Th in which males showed higher values than females.Conclusions:These results stress the complexity of sex and genotype interactions controlling Ca.Th and%PoV,as the skeletal sexual dimorphism was dependent on the genetic background.This study also shows that the CC population is a powerful tool for establishing the genetic effect on these traits.

BMP-IHH-mediated interplay between mesenchymal stem cells and osteoclasts supports calvarial bone homeostasis and repair

Calvarial bones are connected by fibrous sutures. These sutures provide a niche environment that includes mesenchymal stem cells(MSCs), osteoblasts, and osteoclasts, which help maintain calvarial bone homeostasis and repair. Abnormal function of osteogenic cells or diminished MSCs within the cranial suture can lead to skull defects, such as craniosynostosis. Despite the important function of each of these cell types within the cranial suture, we have limited knowledge about the role that crosstalk between them may play in regulating calvarial bone homeostasis and injury repair. Here we show that suture MSCs give rise to osteoprogenitors that show active bone morphogenetic protein(BMP) signalling and depend on BMP-mediated Indian hedgehog(IHH) signalling to balance osteogenesis and osteoclastogenesis activity. IHH signalling and receptor activator of nuclear factor kappa-Β ligand(RANKL) may function synergistically to promote the differentiation and resorption activity of osteoclasts. Loss of Bmpr1a in MSCs leads to downregulation of hedgehog(Hh) signalling and diminished cranial sutures. Significantly, activation of Hh signalling partially restores suture morphology in Bmpr1a mutant mice, suggesting the functional importance of BMP-mediated Hh signalling in regulating suture tissue homeostasis. Furthermore, there is an increased number of CD200+ cells in Bmpr1a mutant mice, which may also contribute to the inhibited osteoclast activity in the sutures of mutant mice. Finally, suture MSCs require BMPmediated Hh signalling during the repair of calvarial bone defects after injury. Collectively, our studies reveal the molecular and cellular mechanisms governing cell–cell interactions within the cranial suture that regulate calvarial bone homeostasis and repair.

Mesoporous Bioglass/Silk Fibroin Scaffolds as a Drug Delivery System: Fabrication, Drug Loading and Release in vitro and Repair Calvarial Defects in vivo

The potential of combining bioactive glass（MBG） and silk fibroin（SF） together as a new drug delivery system was evaluated. The three-dimensional porous scaffolds were selected as the form of SF, and sol-gel method was adopted to fabricate MBG in this study. The characteristic of the synthesized material was measured by transmission electron microscopy and scanning electron microscopy. In vitro evaluation of drug delivery was carried out in terms of drug loading and drug release. And aspirin was chosen as the drug for scaffolds to carry out in vitro tests and repair BALB/C mice calvarial defects. Bone formation was examined by microcomputed tomography. The experimental results show that MBG/silk scaffolds have better physiochemical properties compared with silk scaffolds. In comparison to pure silk scaffolds, MBG/silk scaffolds enhance the drug loading efficiency, release rate in vitro and promote bone regeneration in vivo. Thus we conclude that MBG/silk scaffold is a more efficient drug delivery system than pure silk scaffolds.

Selective serotonin re-uptake inhibitor sertraline inhibits bone healing in a calvarial defect model

Bone wound healing is a highly dynamic and precisely controlled process through which damaged bone undergoes repair and complete regeneration. External factors can alter this process, leading to delayed or failed bone wound healing. The findings of recent studies suggest that the use of selective serotonin reuptake inhibitors(SSRIs) can reduce bone mass, precipitate osteoporotic fractures and increase the rate of dental implant failure. With 10% of Americans prescribed antidepressants, the potential of SSRIs to impair bone healing may adversely affect millions of patients’ ability to heal after sustaining trauma. Here, we investigate the effect of the SSRI sertraline on bone healing through pre-treatment with(10 mg·kg-1sertraline in drinking water, n = 26) or without(control, n = 30) SSRI followed by the creation of a 5-mm calvarial defect. Animals were randomized into three surgical groups:(a) empty/sham,(b) implanted with a DermaMatrix scaffold soak-loaded with sterile PBS or(c) DermaMatrix soak-loaded with542.5 ng BMP2. SSRI exposure continued until sacrifice in the exposed groups at 4 weeks after surgery. Sertraline exposure resulted in decreased bone healing with significant decreases in trabecular thickness, trabecular number and osteoclast dysfunction while significantly increasing mature collagen fiber formation. These findings indicate that sertraline exposure can impair bone wound healing through disruption of bone repair and regeneration while promoting or defaulting to scar formation within the defect site.

Evaluation of Bone Regeneration of Simvastatin Loaded Chitosan Nanofiber Membranes in Rodent Calvarial Defects

Chitosan nanofiber membranes have been known to have a high degree of biocompatibility and support new bone formation with controllable biodegradation. The surface area of these membranes may allow them to serve as local delivery carriers for different biologic mediators. Simvastatin, a drug commonly used for lowering cholesterol, has demonstrated promising bone regenerative capability. The aim of this study was to evaluate simvastatin loaded chitosan nanofiber membranes for guided bone regeneration (GBR) applications and their ability to enhance bone formation in rat calvarial defects. Nanofibrous chitosan membranes with random fiber orientation were fabricated by electrospinning technique and loaded with 0.25 mg of simvastatin under sterile conditions. One membrane was implanted subperiosteally to cover an 8 mm diameter critical size calvarial defect. Two groups: 1) Control: non-loaded chitosan membranes;2) Experimental: chitosan membranes loaded with 0.25 mg of simvastatin were evaluated histologically and via micro-computed tomography (micro-CT) for bone formation at 4 and 8 weeks time points (n = 5/group per time point). Both groups exhibited good biocompatibility with only mild or moderate inflammatory response during the healing process. Histologic and micro-CT evaluations confirmed bone formation in calvarial defects as early as 4 weeks using control and experimental membranes. In addition, newly-formed bony bridges consolidating calvarial defects histologically along with partial radiographic defect coverage were observed at 8 weeks in both groups. Although control and experimental groups demonstrated no significant statistical differences in results of bone formation, biodegradable chitosan nanofiber membranes loaded with simvastatin showed a promising regenerative potential as a barrier material for guided bone regeneration applications.

Repair of critical size rat calvarial defects using endometrial-derived stem cells embedded within gelatin/apatite nanocomposite scaffold

Human stromal stem cells derived from endometrium (hESCs) are a type of multipotent stromal cells of the proven ability to differentiate into osteogenic lineage. Thus, it was suggested that these cells may be used to repair skeletal defects. In this study, Human ESCs were extracted from female endometrium and harvested. Biomimetic gelatin/apatite (Gel/Ap) scaffolds with and without harvested cells were implanted in a Critical size calvarial defects in the cranial bone of adult male rat. To CT-Scan and Histological studies were performed to investigate the level of bone formation after 8 weeks of surgery. Results confirmed that the treated defects with the bare and hESCs grafted Gel/Ap scaffold showed significant bone formation and maturation in comparison with the control group.

Bone morphogenetic protein-9 effectively induces osteogenic differentiation of reversibly immortalized calvarial mesenchymal progenitor cells

Critical-sized craniofacial defect repair represents a significant challenge to reconstructive surgeons.Many strategies have been employed in an effort to achieve both a functionally and cosmetically acceptable outcome.Bone morphogenetic proteins(BMPs)provide a robust osteoinductive cue to stimulate bony growth and remodeling.Previous studies have suggested that the BMP-9 isoform is particularly effective in promoting osteogenic differentiation of mesenchymal progenitor cells.The aim of this study is to characterize the osteogenic capacity of BMP-9 on calvarial mesenchymal progenitor cell differentiation.Reversibly immortalized murine calvarial progenitor cells(iCALs)were infected with adenoviral vectors encoding BMP-9 or GFP and assessed for early and late stages of osteogenic differentiation in vitro and for osteogenic differentiation via in vivo stem cell implantation studies.Significant elevations in alkaline phosphatase(ALP)activity,osteocalcin(OCN)mRNA transcription,osteopontin(OPN)protein expression,and matrix mineralization were detected in BMP-treated cells compared to control.Specifically,ALP activity was elevated on days 3,7,9,11,and 13 post-infection and OCN mRNA expression was elevated on days 8,10,and 14 in treated cells.Additionally,treatment groups demonstrated increased OPN protein expression on day 10 and matrix mineralization on day 14 post-infection relative to control groups.BMP-9 also facilitated the formation of new bone in vivo as detailed by gross,microcomputed tomography,and histological analyses.Therefore,we concluded that BMP-9 significantly stimulates osteogenic differentiation in iCALs,and should be considered an effective agent for calvarial tissue regeneration.

Calvarial defects and Dandy-Walker malformation in association withneurofibromatosis type 1

Neurofibromatosis type 1(NF1)is an autosomal dominant disorder in which the nerve tissue grows tumors that may be benign and may cause serious damage by compressing nerves and other tissues.The NF1 gene

Assessment of necessity of neuronavigation in localization of calvarial extra-axial lesions in the setting of limited resources

Background:Neuronavigation is a very beneficial tool in modern neurosurgical practice.However,the neuronavigation is not available in most of the hospitals in our country raising the question about its importance in localizing the calvarial extra-axial lesions and to what extent it is safe to operate without it.Methods:We studied twenty patients with calvarial extra-axial lesions who underwent surgical interventions.All lesions were preoperatively located with both neuronavigation and the usual linear measurements.Both methods were compared regarding the time consumed to localize the tumor and the accuracy of each method to anticipate the actual center of the tumor.Results:The mean error of distance between the planned center of the tumor and the actual was 6.50±1.762 mm in conventional method,whereas the error was 3.85±1.309 mm in IGS method.Much more time was consumed during the neuronavigation method including booting,registration,and positioning.A statistically significant difference was found between the mean time passed in the conventional method and IGS method(2.05±0.826,24.90±1.334,respectively),P-value<0.001.Conclusion:In the setting of limited resources,the linear measurement localization method seems to have an accepted accuracy in the localization of calvarial extra-axial lesions and it saves more time than neuronavigation method.

Metastases Extracranial Mimicking Meningioma: A Case Report

Background: Skull vault lesions are rare and represent 1% - 2% of all bone masses. Most cerebral metastases are the intra axial tumors, whereas extra-axial masses mimicking meningioma are extremely rare. Case presentation: A 35-year-old woman with a history of mastectomy left breast cancer 5 years below radiotherapy was referred to the neurosurgery department with a parietal extra-axial mass parietal evolving for one year. CT scan with Magnetic resonance imaging revealed an extra-axial tumor with lysis bone. A craniotomy was performed to remove the mass that was located extra-axial. Histopathological examination revealed metastasis. Conclusions: Lesion skull vaults are rare but they should be considered in the differential diagnosis of intraosseous meningioma lesions. In this report, we discuss the clinical aspects of cases we observed, in which the metastasis bone was found thanks to the histological examination of a calvarial mass after surgery.

Effect of clopidogrel in bone healing-experimental study in rabbits

BACKGROUND Clopidogrel is a widely prescribed drug for prevention of myocardial infarction and stroke in patients at risk.It inhibits thrombus formation via inhibition of the P2Y12 purinergic receptor on platelets,which is important in their activation by ADP.However,the P2Y12 receptor has also been found to be expressed in both osteoblasts and osteoclasts.Accumulated evidence suggests that purinergic receptors regulate important functions of bone turnover.Previous studies on the effect of clopidogrel on bone metabolism indicated potential harmful effects,but their results remain conflicting.Thus,clopidogrel treatment may affect bone healing,but it has not yet been studied.AIM To evaluate if continuous perioperative clopidogrel treatment has any negative effect on bone healing in the rabbit calvarial defect model.METHODS Sixteen male white New Zealand rabbits were randomly assigned in two groups:One group received daily 3 mg/kg of clopidogrel per os and the other group received the vehicle alone for a week prior to the surgical procedures;the treatments were continued for another 6 wk postoperatively.The surgical procedures included generation of two circular calvarial defects 11 mm in diameter in every animal.After the 6-wk period of healing,postmortem radiographic and histomorphometric evaluation of the defects was performed.RESULTS Both the surgical procedures and the postoperative period were uneventful and well tolerated by all the animals,without any surgical wound dehiscence,signs of infection or other complication.New bone was formed either inwards from the defect margins or in the central portion of the defect as separated bony islets.While defect healing was still incomplete in both groups,the clopidogrel group had significantly improved radiographic healing scores.Moreover,the histomorphometric analysis showed that bone regeneration(%)was 28.07±7.7 for the clopidogrel group and 19.47±4.9 for the control group,showing a statistically significant difference between them(P=0.018).Statistically significant difference was also found in the defect bridging(%),i.e.72.17±21.2 for the clopidogrel group and 41.17±8.5 for the control group,respectively(P=0.004),whereas there was no statistical difference in bone tissue density between the groups.CONCLUSION Our results indicate that maintenance of perioperative clopidogrel treatment does not negatively affect bone healing but rather promotes it.Further research is needed in order to find useful applications of this finding.

ALP Induction by β－glycerophosphate during the Nonmineralization Phase In Vitro

β-GP influences on rat osteoblast development at the early period of culture i.e , the non-mineralization phase, and changes with the different cell passages were investigated. Alkaline phosphatase (ALP) was chosen as a main object. Northern blot analysis revealed up to two-fold increase in the steady statelevel of ALP mRNA after stimulation of rat osteoblast with 10 mM β-GP- Likewise, 10 mM β-GP induced a 10─30 % increase in ALP activity (P< 0. 01) of early passages (1 to 4), but not of later passages (5 to 6). The β-GP induced increase in ALP activity was totally inhibited by the protein synthesis inhibitor, cycloheximide (50 μM).β- GP stimulation was found to be without effect on cell proliferation measured as 3H-thymidine incorporation. It is concluded that β-GP has no effect on proliferation but induces an increase in both mRNA level and activity of ALP in the non-mineralization phase of cultures of fetal rat calvarial cells , which lasts for several passages but will disappear in older cultures.

Differentiation of Osteoblast in vitro Is Regulated by Progesterone

The regulation of cellular differentiation by progesterone in fetal rat calvarial osteoblasts was investigated. Our results showed that cells cultured in the presence of progesterone had a 7% increase in the alkaline phosphatase activity when compared to untreated cells. The concentration of osteocalcin in the conditioned medium from progesterone treated osteoblasts was 28% higher than that of untreated controls. In addition,administration of progesterone significantly enhanced the number and area of bone nodules. In conclusion, progesterone stimulates the differentiation of fetal rat calvarial osteoblastic cells in vitro

Tissue Engineered Osteogenesis in Bone Defects by Homologous Osteoblasts Loaded on Sterile Bioresorbable Coral Scaffold in Rabbits

Objectives: This study explores feasibility of tissue-engineered osteogenesis using sterile coral implants loaded with homologous osteoblasts to repair bone defects. Study Design: A unilateral 4 mm transverse dis- continuity defect was produced approximately mid-way along left radius of young female rabbits using ro- tary diamond disc under continuous saline irrigation and stabilised with autoclaved steel miniplate and screws. The defect was then fitted with sterile bioresorbable coral implant loaded with homologous neonatal calvarial osteoblasts or control implants without osteoblasts. All animals underwent radiography immedi- ately post-operative, at weekly intervals for four weeks and at fortnightly intervals thereafter. Operated bones were histologically evaluated for osteogenesis at 12 weeks. Results: Findings demonstrate osteogenesis and complete repair of bioresorbable coral implant by homologous osteoblasts loaded on coral scaffold. Conclu- sions: Single stage surgery using this technique to induce osteogenesis and closure of discontinuity bone de- fects including palatal clefts and peripheral reduction of large craniofacial defects might prove better thera- peutic modality than autologous bone grafting or tissue distraction osteogenesis.

Comparative Evaluation of Ozone Treatment in Critical Size Bone Defects Reconstructed with Alloplastic Bone Grafts

The purpose of this study was to investigate osteogenesis promoted by osteoconductive properties of bone grafting materials and the histopathological effects of ozone on osteogenesis. In total, 56 Winstar rats were equally divided into 4 groups. In control group, calvarial bone defect was created in 14 rats. For second group, 8 mm calvarial bone defect with ozone treatment was applied in 14 rats. For third group, an alloplastic bone graft was implanted on 8 mm calvarial bone defect. In fourth group, alloplastic bone graft was inserted in calvarial defect and ozone was treated additionally. Seven of the rats were sacrificed at the end of 4th week and the remaining 7 were sacrificed at the end of 8th week of experiment. In the study, the periosteal flaps were removed with a thin periosteal elevator and averagely 0.8 cm diameter-circular full bone defect was created with a specially designed trephine drill. The bone from the calvarial region was fixed in 10% formalin solution. After decalcification, bones were taken for routine paraffin blocking. Sections were stained with Hematoxylin-Eosin and Masson Trichrome. Histopathological findings of 4th and 8th weeks rats showed that best result for new bone formation was observed in graft + ozone treatment. It is concluded that ozone treatment increases the hemostasis in graft region, induces angiogenesis, promotes cell proliferation by preventing infiltration, induces matrix formation by influencing osteoblastic activity and has a positive effect in osteogenesis.

Effect of covalent-binding modes of osteogenic-related peptides with artificial carriers on their biological activities in vivo

Covalent binding between bioactive substances and materials in different ways can significantly improve the bone inductivity and biological activity of bone repair materials.However,there is a lack of systematic understanding of how these binding modes affect biological activities of the active substances.In this study,four kinds of functionalized Multi-walled carbon nanotubes(MWCNTs)were prepared,ensuring the same grafting rate of different functional groups.Subsequently,two kinds of osteogenic-related peptides,bone morphogenetic protein-2 mimicking peptides and osteogenic growth mimicking peptides,were covalently bound to functionalized MWCNTs,ensuring the same molar mass of peptides bound to different functionalized MWCNTs in this process.Then the same amount of functionalized MWC-NTs/Peptides composites were introduced into the scaffolds,and through the ectopic osteogenesis model in rats and calvarial defect model in rabbits,ectopic osteogenesis and bone repair ability of the composites were analyzed.Furthermore,the effects of different covalent binding modes on peptide-induced osteogenesis and bone repair were studied.The results showed that the negative influencing trend of different covalent binding modes of osteogenic-related peptides with artificial carriers on their biological activities was in the order as follows:amide binding(carboxyl)>silane coupling>dopamine bind-ing>amide binding(amino),whose mechanism might be mainly that the covalent binding of peptides with different functional groups resulted in different charges.We believe that the results of this study have important guiding significance for the research and development of bone repair materials covalently bound with bioactive substances.

Exosome-functionalized magnesium-organic framework-based scaffoldswith osteogenic, angiogenic and anti-inflammatory properties foraccelerated bone regeneration

Exosomes derived from human adipose-derived stem cells (hADSCs-Exos) have shown potential as an effectivetherapeutic tool for repairing bone defects. Although metal-organic framework (MOF) scaffolds are promisingstrategies for bone tissue regeneration, their potential use for exosome loading remains unexplored. In this study,motivated by the potential advantages of hADSCs-Exos and Mg-GA MOF, we designed and synthesized anexosome-functionalized cell-free PLGA/Mg-GA MOF (PLGA/Exo-Mg-GA MOF) scaffold, taking using of thebenefits of hADSCs-Exos, Mg2+, and gallic acid (GA) to construct unique nanostructural interfaces to enhanceosteogenic, angiogenic and anti-inflammatory capabilities simultaneously. Our in vitro work demonstrated thebeneficial effects of PLGA/Exo-Mg-GA MOF composite scaffolds on the osteogenic effects in human bonemarrow-derived mesenchymal stem cells (hBMSCs) and angiogenic effects in human umbilical endothelial cells(HUVECs). Slowly released hADSCs-Exos from composite scaffolds were phagocytosed by co-cultured cells,stabilized the bone graft environment, ensured blood supply, promoted osteogenic differentiation, and acceleratedbone reconstruction. Furthermore, our in vivo experiments with rat calvarial defect model showed thatPLGA/Exo-Mg-GA MOF scaffolds promoted new bone formation and satisfactory osseointegration. Overall, weprovide valuable new insights for designing exosome-coated nanocomposite scaffolds with enhanced osteogenesisproperty.

Hyaluronic acid-based hydrogels with tobacco mosaic virus containing cell adhesive peptide induce bone repair in normal and osteoporotic rats

Tobacco mosaic virus(TMV)has been studied as a multi-functional agent for bone tissue engineering.An osteo-inductive effect of wild-type TMV has been reported,as it can significantly enhance the bone differentiation potential of bone marrow stromal cells both on a two-dimensional substrate and in a three-dimensional(3D)hydrogel system.A TMV mutant(TMV-RGD1)was created which featured the adhesion peptide arginyl-glycyl-aspartic acid(RGD),the most common peptide motif responsible for cell adhesion to the extracellular matrix,on the surface of the virus particle to enhance the bio-functionality of the scaffold material.We hypothesised that the incorporation of either wild-type TMV or TMV-RGD1 in the 3D hydrogel scaffold would induce bone healing in critical size defects of the cranial segmental bone.We have previously tested the virus-functionalised scaffolds,in vitro,with a hyaluronic acid-based system as an in-situ hydrogel platform for 3D cell encapsulation,culture,and differentiation.The results of these experiments suggested the potential of the virus-functionalised hydrogel to promote in vitro stem cell differentiation.The hydrogel-forming system we employed was shown to be safe and biocompatible in vivo.Here,we further explored the physiological responses regarding bone regeneration of a calvarial defect in both normal and osteoporotic ovariectomized rat models.Our results,based on histological analysis in both animal models,suggested that both wild-type TMV and TMV-RGD1 functionalised hydrogels could accelerate bone regeneration,without systemic toxicity,evaluated by blood counts.New bone formation was intensified by the incorporation of the RGD-mutant viral particles.This finding increased the potential for use of the rodshaped plant virus as a platform for the addition of powerful biofunctionality for tissue engineering applications.This study was approved by the Ethics Committee on Animal Use of the Zhenjiang Affiliated First People’s Hospital affiliated to Jiangsu University.

Regeneration of critical-sized defects, in a goat model, using a dextrin-based hydrogel associated with granular synthetic bone substitute

The development of injectable bone substitutes(IBS)have obtained great importance in the bone re-generation field,as a strategy to reach hardly accessible defects using minimally invasive techniques and able to fit to irregular topographies.In this scenario,the association of injectable hydrogels and bone graft granules is emerging as a well-established trend.Particularly,in situ forming hydrogels have arisen as a new IBS generation.An in situ forming and injectable dextrin-based hydrogel(HG)was developed,aiming to act as a carrier of granular bone substitutes and bioactive agents.In this work,the HG was associated to a granular bone substitute(Bonelike)and implanted in goat critical-sized calvarial defects(14mm)for 3,6 and 12weeks.The results showed that HG improved the han-dling properties of the Bonelike granules and did not affect its osteoconductive features,neither impairing the bone regener ation process.Human multipotent mesenchymal stromal cells from the umbilical cord,extracellular matrix hydrolysates and the pro-angiogenic peptide LLKKK18 were also combined with the IBS.These bioactive agents did not enhance the new bone formation significantly under the conditions tested,according to micro-computed tomography and histological analysis.

Next-generation resorbable polymer scaffolds with surface-precipitated calcium phosphate coatings

Next-generation synthetic bone graft therapies will most likely be composed of resorbable polymers in combination with bioactive components.In this article,we continue our exploration of E1001(1k),a tyrosine-derived polycarbonate,as an orthopedic implant material.Specifically,we use E1001(1k),which is degradable,nontoxic,and osteoconductive,to fabricate porous bone regeneration scaffolds that were enhanced by two different types of calcium phosphate(CP)coatings:in one case,pure dicalcium phosphate dihydrate was precipitated on the scaffold surface and throughout its porous structure(E1001(1k)+CP).In the other case,bone matrix minerals(BMM)such as zinc,manganese and fluoride were co-precipitated within the dicalcium phosphate dihydrate coating(E1001(1k)+BMM).These scaffold compositions were compared against each other and against ChronOS(Synthes USA,West Chester,PA,USA),a clinically used bone graft substitute(BGS),which served as the positive control in our experimental design.This BGS is composed of poly(lactide co-e-caprolactone)and beta-tricalcium phosphate.We used the established rabbit calvaria critical-sized defect model to determine bone regeneration within the defect for each of the three scaffold compositions.New bone formation was determined after 2,4,6,8 and 12 weeks by micro-computerized tomography(mCT)and histology.The experimental tyrosine-derived polycarbonate,enhanced with dicalcium phosphate dihydrate,E1001(1k)+CP,supported significant bone formation within the defects and was superior to the same scaffold containing a mix of BMM,E1001(1k)+BMM.The comparison with the commercially available BGS was complicated by the large variability in bone formation observed for the laboratory preparations of E1001(1k)scaffolds.At all time points,there was a trend for E1001(1k)+CP to be superior to the commercial BGS.However,only at the 6-week time point did this trend reach statistical significance.Detailed analysis of the μCT data suggested an increase in bone formation from 2 through 12 weeks in implant sites treated with E1001(1k)+CP.At 2 and 4 weeks post-implantation,bone formation occurred at the interface where the E1001(1k)+CP scaffold was in contact with the bone borders of the implant site.Thereafter,during weeks 6,8 and 12 bone formation progressed throughout the E1001(1k)+CP test implants.This trend was not observed with E1001(1k)+BMM scaffolds or the clinically used BGS.Our results suggest that E1001(1k)+CP should be tested further for osteoregenerative applications.