Osmolarity-Induced Altered Intracellular Molecular Crowding Drives Osteoarthritis Pathology.

Osteoarthritis (OA) is a multifactorial degenerative joint disease of which the underlying mechanisms are yet to be fully understood. At the molecular level, multiple factors including altered signaling pathways, epigenetics, metabolic imbalance, extracellular matrix degradation, production of matrix metalloproteinases, and inflammatory cytokines, are known to play a detrimental role in OA. However, these factors do not initiate OA, but are mediators or consequences of the disease, while many other factors causing the etiology of OA are still unknown. Here, it is revealed that microenvironmental osmolarity can induce and reverse osteoarthritis-related behavior of chondrocytes via altered intracellular molecular crowding, which represents a previously unknown mechanism underlying OA pathophysiology. Decreased intracellular crowding is associated with increased sensitivity to proinflammatory triggers and decreased responsiveness to anabolic stimuli. OA-induced lowered intracellular molecular crowding could be renormalized via exposure to higher extracellular osmolarity such as those found in healthy joints, which reverse OA chondrocyte's sensitivity to catabolic stimuli as well as its glycolytic metabolism.

The effect of surface roughening on the success of orthodontic mini-implants: A systematic review and meta-analysis.

INTRODUCTION: Orthodontic mini-implants are a widely accepted treatment modality in orthodontics; however, the failure rate is moderately high. Surface roughening is the golden standard in conventional oral implantology, and this may prove beneficial for orthodontic mini-implants as well. The objective of this systematic review is to assess the effect of surface roughening on the success rate of orthodontic mini-implants in both adolescent and adult patients undergoing orthodontic treatment. METHODS: Randomized studies comparing the success of surface-roughened and smooth, machined-surface orthodontic mini-implants were included. A literature search was conducted for 6 electronic databases (Pubmed/Medline, Embase, Cochrane, CINAHL, Web of Science, and Scopus), Clinical trial registry (https://www. CLINICALTRIALS: gov), and grey literature (Google Scholar). A manual search of the reference lists of included studies was performed. Two authors independently performed the screening, data extraction, risk of bias, and quality assessments. The risk of bias was assessed with the Cochrane risk-of-bias 2.0 Tool. Data were synthesized using a random effect model meta-analysis presented as a forest plot. The certainty in the body of evidence was assessed using the Grading of Recommendations Assessment, Development, and Evaluation tool. RESULTS: A total of 4226 unique records were screened, and 6 of these were included in the quantitative analysis. Four additional articles were selected for a secondary outcome. A total of 364 orthodontic mini-implants were included in the primary outcome analysis. There was no statistically significant effect of surface roughening on the success of orthodontic mini-implants (odds ratio = 0.63 favoring roughened orthodontic mini-implants; 95% confidence interval, 0.35-1.14). The secondary outcome (ie, the overall failure rate of roughened orthodontic mini-implants) was 6% based on studies with high heterogeneity. Limitations of this study were the risk of bias, study imprecision, and possible publication bias, leading to a very low certainty in the body of evidence. CONCLUSIONS: There is very low-quality evidence that there is no statistically significant effect of surface roughening on the success of orthodontic mini-implants in humans. The overall failure rate of surface-roughened orthodontic mini-implants was 6%. FUNDING: No funding was received for this review. REGISTRATION: This study was preregistered in the Prospective Register of Systematic Reviews (CRD42022371830).

Bifunctional bone substitute materials for bone defect treatment after bone tumor resection.

Aggressive benign, malignant and metastatic bone tumors can greatly decrease the quality of patients' lives and even lead to substantial mortality. Several clinical therapeutic strategies have been developed to treat bone tumors, including preoperative chemotherapy, surgical resection of the tumor tissue, and subsequent systemic chemo- or radiotherapy. However, those strategies are associated with inevitable drawbacks, such as severe side effects, substantial local tumor recurrence, and difficult-to-treat bone defects after tumor resection. To overcome these shortcomings and achieve satisfactory clinical outcomes, advanced bifunctional biomaterials which simultaneously promote bone regeneration and combat bone tumor growth are increasingly advocated. These bifunctional bone substitute materials fill bone defects following bone tumor resection and subsequently exert local anticancer effects. Here we describe various types of the most prevalent bone tumors and provide an overview of common treatment options. Subsequently, we review current progress regarding the development of bifunctional bone substitute materials combining osteogenic and anticancer efficacy. To this end, we categorize these biomaterials based on their anticancer mechanism deriving from i) intrinsic biomaterial properties, ii) local drug release of anticancer agents, and iii) oxidative stress-inducing and iv) hyperthermia-inducing biomaterials. Consequently, this review offers researchers, surgeons and oncologists an up-to-date overview of our current knowledge on bone tumors, their treatment options, and design of advanced bifunctional biomaterials with strong potential for clinical application in oncological orthopedics.

Lateral Bone Augmentation Using a Three-Dimensional-Printed Polymeric Chamber to Compare Biomaterials.

The aim of this study was to test the suitability of calcium phosphate cement mixed with poly(lactic-co-glycolic acid) (CPC-PLGA) microparticles into a ring-shaped polymeric space-maintaining device as bone graft material for lateral bone augmentation. Therefore, the bone chambers were installed on the lateral portion of the anterior region of the mandibular body of mini-pigs. Chambers were filled with either CPC-PLGA or BioOss® particles for comparison and left for 4 and 12 weeks. Histology and histomorphometry were used to obtain temporal insight in material degradation and bone formation. Results indicated that between 4 and 12 weeks of implantation, a significant degradation of the CPC-PLGA (from 75.1% to 23.1%), as well as BioOss material, occurred (from 40.6% to 14.4%). Degradation of both materials was associated with the presence of macrophage-like and osteoclast-like cells. Furthermore, a significant increase in bone formation occurred between 4 and 12 weeks for the CPC-PLGA (from 0.1% to 7.2%), as well as BioOss material (from 8.3% to 23.3%). Statistical analysis showed that bone formation had progressed significantly better using BioOss compared to CPC-PLGA (p < 0.05). In conclusion, this mini-pig study showed that CPC-PLGA does not stimulate lateral bone augmentation using a bone chamber device. Both treatments failed to achieve "clinically" meaningful alveolar ridge augmentation.

Influence of bisphosphonate treatment on bone substitute performance in osteoporotic conditions.

OBJECTIVE: Considering the elevated number of osteoporotic patients in need of bone graft procedures, we here evaluated the effect of alendronate (ALN) treatment on the regeneration of bone defects in osteoporotic rats. Bone formation was histologically and histomorphometrically assessed in rat femoral condyle bone defects filled with bone graft (Bio-Oss®) or left empty. METHODS: Male Wistar rats were induced osteoporotic through orchidectomy (ORX) and SHAM-operated. The animals were divided into three groups: osteoporotic (ORX), osteoporotic treated with ALN (ORX + ALN) and healthy (SHAM). Six weeks after ORX or SHAM surgeries, bone defects were created bilaterally in femoral condyles; one defect was filled with Bio-Oss® and the other one left empty. Bone regeneration within the defects was analyzed by histology and histomorphometry after 4 and 12 weeks. RESULTS: Histological samples showed new bone surrounding Bio-Oss® particles from week 4 onward in all three groups. At week 12, the data further showed that ALN treatment of osteoporotic animals enhanced bone formation to a 10-fold increase compared to non-treated osteoporotic control. Bio-Oss® filling of the defects promoted bone formation at both implantation periods compared to empty controls. CONCLUSION: Our histological and histomorphometric results demonstrate that the enteral administration of alendronate under osteoporotic bone conditions leverages bone defect regeneration to a level comparable to that in healthy bone. Additionally, Bio-Oss® is an effective bone substitute, increasing bone formation, and acting as an osteoconductive scaffold guiding bone growth in both healthy and osteoporotic bone conditions. SIGNIFICANCE: Based on the results of this study, enteral use of ALN mitigates adverse effects of an osteoporotic condition on bone defect regeneration.

Comparison of Osteogenic Capacity and Osteoinduction of Adipose Tissue-Derived Cell Populations.

Stromal vascular fraction (SVF) is the primary isolate obtained after enzymatic digestion of adipose tissue that contains various cell types. Its successful application for cell-based construct preparation in an intra-operative setting for clinical bone augmentation and regeneration has been previously reported. However, the performance of SVF-based constructs compared with traditional ex vivo expanded adipose tissue-derived mesenchymal stromal cells (ATMSCs) remains unclear and direct comparative analyses are scarce. Consequently, we here aimed at comparing the in vitro osteogenic differentiation capacity of donor-matched SVF versus ATMSCs as well as their osteoinductive capacity. Human adipose tissue from nine different donors was used to isolate SVF, which was further purified via plastic-adherence to obtain donor-matched ATMSCs. Both cell populations were immunophenotypically characterized for mesenchymal stromal cell, endothelial, and hematopoietic markers after isolation and immunocytochemical staining was used to identify different cell types during prolonged cell culture. Based on normalization using plastic-adherence fraction determination, SVF and ATMSCs were seeded and cultured in osteogenic differentiation medium for 28 days. Further, SVF and ATMSCs were seeded onto devitalized bovine bone granules and subcutaneously implanted into nude mice. After 42 days of implantation, granules were retrieved, histologically processed, and stained with hematoxylin and eosin (HE) to assess ectopic bone formation. The ATMSCs were shown to be a homogenous cell population during cell culture, whereas SVF cultures consisted of multiple cell types. All donor-matched comparisons showed either accelerated or stronger mineralization for SVF cultures in vitro. However, neither SVF nor ATMSCs loaded on devitalized bone granules induced ectopic bone formation on subcutaneous implantation, as opposed to control granules loaded with bone morphogenetic protein-2 (BMP-2), which triggered ectopic bone formation with 100% incidence. Despite the observed lack of osteoinduction, our findings provide important in vitro evidence on the osteogenic superiority of intra-operatively available SVF as compared with donor-matched ATMSCs. Consequently, further studies should focus on optimizing the efficacy of these cell populations for implementation in orthotopic bone fracture or defect treatment.

Repair of complex ovine segmental mandibulectomy utilizing customized tissue engineered bony flaps.

Craniofacial defects require a treatment approach that provides both robust tissues to withstand the forces of mastication and high geometric fidelity that allows restoration of facial architecture. When the surrounding soft tissue is compromised either through lack of quantity (insufficient soft tissue to enclose a graft) or quality (insufficient vascularity or inducible cells), a vascularized construct is needed for reconstruction. Tissue engineering using customized 3D printed bioreactors enables the generation of mechanically robust, vascularized bony tissues of the desired geometry. While this approach has been shown to be effective when utilized for reconstruction of non-load bearing ovine angular defects and partial segmental defects, the two-stage approach to mandibular reconstruction requires testing in a large, load-bearing defect. In this study, 5 sheep underwent bioreactor implantation and the creation of a load-bearing mandibular defect. Two bioreactor geometries were tested: a larger complex bioreactor with a central groove, and a smaller rectangular bioreactor that were filled with a mix of xenograft and autograft (initial bone volume/total volume BV/TV of 31.8 ± 1.6%). At transfer, the tissues generated within large and small bioreactors were composed of a mix of lamellar and woven bone and had BV/TV of 55.3 ± 2.6% and 59.2 ± 6.3%, respectively. After transfer of the large bioreactors to the mandibular defect, the bioreactor tissues continued to remodel, reaching a final BV/TV of 64.5 ± 6.2%. Despite recalcitrant infections, viable osteoblasts were seen within the transferred tissues to the mandibular site at the end of the study, suggesting that a vascularized customized bony flap is a potentially effective reconstructive strategy when combined with an optimal stabilization strategy and local antibiotic delivery prior to development of a deep-seated infection.

Fast Degradable Calcium Phosphate Cement for Maxillofacial Bone Regeneration.

The aim of this preclinical study was to test the applicability of calcium phosphate cement (CPC)-poly(lactic-co-glycolic acid) (PLGA)-carboxymethylcellulose (CMC) as a bone substitute material for guided bone regeneration (GBR) procedures in a clinically relevant mandibular defect model in minipigs. In the study, a predicate device (i.e., BioOss®) was included for comparison. Critical-sized circular mandibular bone defects were created and filled with either CPC-PLGA-CMC without coverage with a GBR membrane or BioOss covered with a GBR membrane and left to heal for 4 and 12 weeks to obtain temporal insight in material degradation and bone formation. Bone formation increased significantly for both CPC-PLGA-CMC and BioOss with increasing implantation time. Further, no significant differences were found for bone formation at either 4 or 12 weeks between CPC-PLGA-CMC and BioOss. Finally, bone substitute material degradation increased significantly for both CPC-PLGA-CMC and BioOss from 4 to 12 weeks of implantation, showing the highest degradation for CPC-PLGA-CMC (∼85%) compared to BioOss (∼12%). In conclusion, this minipig study showed that CPC-PLGA-CMC can be used as a bone-grafting material and stimulates bone regeneration to a comparable extent as with BioOss particles. Importantly, CPC-PLGA-CMC degrades faster compared to BioOss, is easier to apply into a bone defect, and does not need the use of an additional GBR membrane. Consequently, the data support the further investigation of CPC-PLGA-CMC in human clinical trials. Impact statement Guided bone regeneration (GBR) is a frequently used dental surgical technique to regenerate the alveolar ridge to allow stable implant installation. However, stabilization of the GBR membrane and avoidance of bone graft movement remain a challenge. Consequently, there is need for the development of alternative materials to be used in GBR procedures that are easier to apply and induce predictable bone regeneration. In this minipig study, we focused on the applicability of calcium phosphate cement-poly(lactic-co-glycolic acid)-carboxymethylcellulose as an alternative bone substitute material for GBR procedures without the need of an additional GBR membrane.

Cleaved SPP1-rich extracellular vesicles from osteoclasts promote bone regeneration via TGFß1/SMAD3 signaling.

Bone remodeling is a tightly coupled process between bone forming osteoblasts (OBs) and bone resorbing osteoclasts (OCs) to maintain bone architecture and systemic mineral homeostasis throughout life. However, the mechanisms responsible for the coupling between OCs and OBs have not been fully elucidated. Herein, we first validate that secreted extracellular vesicles by osteoclasts (OC-EVs) promote osteogenic differentiation of mesenchymal stem cells (MSCs) and further demonstrate the efficacy of osteoclasts and their secreted EVs in treating tibial bone defects. Furthermore, we show that OC-EVs contain several osteogenesis-promoting proteins as cargo. By employing proteomic and functional analysis, we reveal that mature osteoclasts secrete thrombin cleaved phosphoprotein 1 (SPP1) through extracellular vesicles which triggers MSCs osteogenic differentiation into OBs by activating Transforming Growth Factor ß1 (TGFß1) and Smad family member 3 (SMAD3) signaling. In conclusion, our findings prove an important role of SPP1, present as cargo in OC-derived EVs, in signaling to MSCs and driving their differentiation into OBs. This biological mechanism implies a paradigm shift regarding the role of osteoclasts and their signaling toward the treatment of skeletal disorders which require bone formation.

Race for Applicable Antimicrobial Dental Implant Surfaces to Fight Biofilm-Related Disease: Advancing in Laboratorial Studies vs Stagnation in Clinical Application.

Across years, potential strategies to fight peri-implantitis have been notoriously explored through the antimicrobial coating implant surfaces capable of interfering with the bacterial adhesion process. However, although experimental studies have significantly advanced, no product has been marketed so far. For science to reach the society, the commercialization of research outcomes is necessary to provide real advancement in the biomedical field. Therefore, the aim of this study was to investigate the challenges involved in the development of antimicrobial dental implant surfaces to fight peri-implantitis, through a systematic search. Research articles reporting antimicrobial dental implant surfaces were identified by searching PubMed, Scopus, Web of Science, The Cochrane Library, Embase, and System of Information on Grey Literature in Europe, between 2008 and 2020. A total of 1778 studies were included for quality assessment and the review. An impressive number of 1655 articles (93,1%) comprised in vitro studies, whereas 123 articles refer to in vivo investigations. From those 123, 102 refer to animal studies and only 21 articles were published on the clinical performance of antibacterial dental implant surfaces. The purpose of animal studies is to test how safe and effective new treatments are before they are tested in people. Therefore, the discrepancy between the number of published studies clearly reveals that preclinical investigations still come up against several challenges to overcome before moving forward to a clinical setting. Additionally, researchers need to recognize that the complex journey from lab to market requires more than a great idea and resources to develop a commercial invention; research teams must possess the skills necessary to commercialize an invention.

Tailoring Cu2+-loaded electrospun membranes with antibacterial ability for guided bone regeneration.

Copper (Cu)-loaded electrospun membranes were tailored for guided bone regeneration (GBR), targeting the stimulation of innate cells active in bone growth and the prevention of bacterial infections. Functional GBR membranes were produced via an electrospinning set-up using a silk-based solution associated with polyethylene oxide (Silk/PEO - control). Experimental groups were loaded with copper oxide using varying weight percentages (0.05 % to 1 % of CuO). The morphological, structural, chemical, and mechanical properties of membranes were evaluated. Direct and indirect in vitro cytocompatibility experiments were performed with primary human bone mesenchymal stem cells and primary human umbilical vein endothelial cells. The antibacterial potential of membranes was tested with Staphylococcus aureus and Fusobacterium nucleatum biofilm. CuO was successfully incorporated into membranes as clusters without compromising their mechanical properties for clinical applicability. Increased Cu concentrations generated membranes with thinner nanofibers, greater pore areas, and stronger antimicrobial effect (p < 0.01). Cu2+ ion was released from the nanofiber membranes during 1 week, showing higher release in acidic conditions. CuO 0.1 % and CuO 0.05 % membranes were able to support and stimulate cell adhesion and proliferation (p < 0.05), and favor angiogenic responses of vascular cells. In addition, detailed quantitative and qualitative analysis determined that amount of the attached biofilm was reduced on the tailored functional Cu2+-loaded GBR membrane. Importantly, these qualities represent a valuable strategy to improve the bone regeneration process and diminish the risk of bacterial infections.

Nanoindentation and nano-scratching of hydroxyapatite coatings for resorbable magnesium alloy bone implant applications.

The corrosion rate of Mg alloys is currently too high for viable resorbable implant applications. One possible solution is to coat the alloy with a hydroxyapatite (HA) layer to slow the corrosion and promote bone growth. As such coatings can be under severe stresses during implant insertion, we present a nano-mechanical and nano-tribological investigation of RF-sputtered HA films on AZ31 Mg alloy substrates. EDX and XRD analysis indicate that as-deposited coatings are amorphous and Ca-deficient whereas rapid thermal annealing results in c-axis orientation and near-stoichiometric composition. Analysis of the nanoindentation data using a thin film model shows that annealing increases the coating's intrinsic hardness (H) and strain at break (H/E) values, from 2.7 GPa to 9.4 GPa and from 0.043 to 0.079, respectively. In addition, despite being rougher, the annealed samples display better wear resistance; a sign that the rapid thermal annealing does not compromise their interfacial strength and that these systems have potential for resorbable bone implant applications.

Copper source determines chemistry and topography of implant coatings to optimally couple cellular responses and antibacterial activity.

Implant-related infections at the early healing period are considered one of the main risk factors in implant failure. Designing coatings that control bacterial adhesion and have cell stimulatory behavior remains a challenging strategy for dental implants. Here, we used plasma electrolytic oxidation (PEO) to produce antimicrobial coatings on commercially pure titanium (cpTi) using bioactive elements (calcium and phosphorus) and different copper (Cu) sources: copper acetate (CuAc), copper sulfate (CuS), and copper oxide (CuO); coatings containing only Ca and P (CaP) served as controls. Cu sources drove differential physical and chemical surface features of PEO coatings, resulting in tailorable release kinetics with a sustained Cu ion release over 10 weeks. The antibacterial effects of Cu-containing coatings were roughness-dependent. CuAc coating exhibited optimal properties in terms of its hydrophilicity, pores density, and limited surface roughness, which provided the most robust antibacterial activity combined with appropriate responses of human primary stem cells and angiogenic cells. Our data indicate that Cu source selection largely determines the functionality of Cu-containing PEO coatings regarding their antibacterial efficacy and cytocompatibility.

Sustained local ionic homeostatic imbalance caused by calcification modulates inflammation to trigger heterotopic ossification.

Heterotopic ossification (HO) is a condition triggered by an injury leading to the formation of mature lamellar bone in extraskeletal soft tissues. Despite being a frequent complication of orthopedic and trauma surgery, brain and spinal injury, the etiology of HO is poorly understood. The aim of this study is to evaluate the hypothesis that a sustained local ionic homeostatic imbalance (SLIHI) created by mineral formation during tissue calcification modulates inflammation to trigger HO. This evaluation also considers the role SLIHI could play for the design of cell-free, drug-free osteoinductive bone graft substitutes. The evaluation contains five main sections. The first section defines relevant concepts in the context of HO and provides a summary of proposed causes of HO. The second section starts with a detailed analysis of the occurrence and involvement of calcification in HO. It is followed by an explanation of the causes of calcification and its consequences. This allows to speculate on the potential chemical modulators of inflammation and triggers of HO. The end of this second section is devoted to in vitro mineralization tests used to predict the ectopic potential of materials. The third section reviews the biological cascade of events occurring during pathological and material-induced HO, and attempts to propose a quantitative timeline of HO formation. The fourth section looks at potential ways to control HO formation, either acting on SLIHI or on inflammation. Chemical, physical, and drug-based approaches are considered. Finally, the evaluation finishes with a critical assessment of the definition of osteoinduction. STATEMENT OF SIGNIFICANCE: The ability to regenerate bone in a spatially controlled and reproducible manner is an essential prerequisite for the treatment of large bone defects. As such, understanding the mechanism leading to heterotopic ossification (HO), a condition triggered by an injury leading to the formation of mature lamellar bone in extraskeletal soft tissues, would be very useful. Unfortunately, the mechanism(s) behind HO is(are) poorly understood. The present study reviews the literature on HO and based on it, proposes that HO can be caused by a combination of inflammation and calcification. This mechanism helps to better understand current strategies to prevent and treat HO. It also shows new opportunities to improve the treatment of bone defects in orthopedic and dental procedures.

Dual-functional porous and cisplatin-loaded polymethylmethacrylate cement for reconstruction of load-bearing bone defect kills bone tumor cells.

Malignant bone tumors are usually treated by resection of tumor tissue followed by filling of the bone defect with bone graft substitutes. Polymethylmethacrylate (PMMA) cement is the most commonly used bone substitute in clinical orthopedics in view of its reliability. However, the dense nature of PMMA renders this biomaterial unsuitable for local delivery of chemotherapeutic drugs to limit the recurrence of bone tumors. Here, we introduce porosity into PMMA cement by adding carboxymethylcellulose (CMC) to facilitate such local delivery of chemotherapeutic drugs, while retaining sufficient mechanical properties for bone reconstruction in load-bearing sites. Our results show that the mechanical strength of PMMA-based cements gradually decreases with increasing CMC content. Upon incorporation of ≥3% CMC, the PMMA-based cements released up to 18% of the loaded cisplatin, in contrast to cements containing lower amounts of CMC which only released less than 2% of the cisplatin over 28 days. This release of cisplatin efficiently killed osteosarcoma cells in vitro and the fraction of dead cells increased to 91.3% at day 7, which confirms the retained chemotherapeutic activity of released cisplatin from these PMMA-based cements. Additionally, tibias filled with PMMA-based cements containing up to 3% of CMC exhibit comparable compressive strengths as compared to intact tibias. In conclusion, we demonstrate that PMMA cements can be rendered therapeutically active by introducing porosity using CMC to allow for release of cisplatin without compromising mechanical properties beyond critical levels. As such, these data suggest that our dual-functional PMMA-based cements represent a viable treatment option for filling bone defects after bone tumor resection in load-bearing sites.

A Mini-Pig Mandibular Defect Model for Evaluation of Craniomaxillofacial Bone Regeneration.

Craniomaxillofacial bone defects represent a clinical challenge in the fields of maxillofacial surgery and (implant) dentistry. Regeneration of these bone defects requires the application of bone graft materials that facilitate new bone formation in a safe, reliable, and predictive manner. In addition to autologous bone graft, several types of (synthetic) bone substitute materials have become clinically available, and still major efforts are focused on improving such bone substitute materials by optimizing their properties. Given the regulatory necessity to evaluate the performance of new bone substitute materials for craniomaxillofacial bone regeneration in a large animal model with similarity to human bone before clinical application, we here describe a mini-pig mandibular bone defect model that allows for the creation of multiple (critical-size) bone defects within the mandibular body of a single animal. As examples of bone substitute materials, we utilize both the clinically used BioOss granules and an experimental calcium phosphate cement for filling the created defects. Regarding the latter, its advantages are the injectable application within the defect site, in which the material rapidly sets, and the tailorable degradation properties via the inclusion of hydrolytically degrading polymeric particles. For both bone substitute materials, we show the suitability of the bone defect model to assess bone regeneration via histology and micro-computed tomography. Impact statement Given the regulatory necessity to evaluate the performance of new bone substitute materials for craniomaxillofacial bone regeneration in a large animal model with similarity to the human bone before clinical application, we here describe a mini-pig mandibular bone defect model that allows for the creation of multiple (critical-size) bone defects within the mandibular body of a single animal that can be used for the evaluation of the bone regenerative capacity of new bone grafting materials as well as tissue-engineered products for alveolar bone regeneration.