Atypical histological presentation of bone regeneration after insertion of cryoprotected allogeneic bone graf / Presentación histológica atípica de la regeneración ósea después de la inserción de un injerto óseo alogénico crioprotegido

Background: To evaluate bone regenerative capacity of cryoprotected corticocancellous allogeneic bone graftperformed in type II and III post-extraction sockets for ridge preservation after twelve weeks in-vivo.Material and Methods: Twenty-seven type II or III bony-walled extraction sockets (mandible and maxilla) wereselected for this study. Following atraumatic tooth-extraction a cryoprotected corticocancellous allogeneic bonegraft material and a resorbable porcine-derived collagen membrane were used for ridge preservation. Duringre-entry surgery at approximately 12 weeks, bone core biopsies were obtained using a 3.2 mm trephine drill andsamples were histologically processed and subjected to qualitative and quantitative histomorphometric analysis.Quantitative data was analyzed using a general linear mixed model with results presented as mean values with thecorresponding 95% confidence interval values. Results: Healing without incident and ridge preservation allowed for the placement of dental implants after 12 weeksin 25 out of the 27 treated socket sites. Analyses yielded an average of ~21.0±7% of old/native bone, ~17±5.5% ofnewly regenerated bone (total of ~38±12.8% for all bone), 0.23±0.14% of new bone presenting with nucleating siteswithin the matrix, ~52±5.12% of soft tissue, and 3.6±2.09% of damaged bone. The average regenerated bone wasstatistically analogous to that of old/native bone (p=0.355). Furthermore, an atypical histological pattern of boneregeneration was observed, with newly formed bone exhibiting “infiltration-like” behavior and with new bone nucle-ating sites observed within the demineralized bone matrix.Conclusions: Cryoprotected corticocancellous allogeneic bone-graft demonstrated osteoconductive, osteoinductive,and osteogenic properties, yielding unique healing patterns which does warrant further investigation.(AU)

Bone Tissue Engineering (BTE) of the Craniofacial Skeleton, Part II: Translational Potential of 3D-Printed Scaffolds for Defect Repair.

Computer-aided design/computer-aided manufacturing and 3-dimensional (3D) printing techniques have revolutionized the approach to bone tissue engineering for the repair of craniomaxillofacial skeletal defects. Ample research has been performed to gain a fundamental understanding of the optimal 3D-printed scaffold design and composition to facilitate appropriate bone formation and healing. Benchtop and preclinical, small animal model testing of 3D-printed bioactive ceramic scaffolds augmented with pharmacological/biological agents have yielded promising results given their potential combined osteogenic and osteoinductive capacity. However, other factors must be evaluated before newly developed constructs may be considered analogous alternatives to the "gold standard" autologous graft for defect repair. More specifically, the 3D-printed bioactive ceramic scaffold's long-term safety profile, biocompatibility, and resorption kinetics must be studied. The ultimate goal is to successfully regenerate bone that is comparable in volume, density, histologic composition, and mechanical strength to that of native bone. In vivo studies of these newly developed bone tissue engineering in translational animal models continue to make strides toward addressing regulatory and clinically relevant topics. These include the use of skeletally immature animal models to address the challenges posed by craniomaxillofacial defect repair in pediatric patients. This manuscript reviews the most recent preclinical animal studies seeking to assess 3D-printed ceramic scaffolds for improved repair of critical-sized craniofacial bony defects.

Loss of Notch signaling in skeletal stem cells enhances bone formation with aging.

Skeletal stem and progenitor cells (SSPCs) perform bone maintenance and repair. With age, they produce fewer osteoblasts and more adipocytes leading to a loss of skeletal integrity. The molecular mechanisms that underlie this detrimental transformation are largely unknown. Single-cell RNA sequencing revealed that Notch signaling becomes elevated in SSPCs during aging. To examine the role of increased Notch activity, we deleted Nicastrin, an essential Notch pathway component, in SSPCs in vivo. Middle-aged conditional knockout mice displayed elevated SSPC osteo-lineage gene expression, increased trabecular bone mass, reduced bone marrow adiposity, and enhanced bone repair. Thus, Notch regulates SSPC cell fate decisions, and moderating Notch signaling ameliorates the skeletal aging phenotype, increasing bone mass even beyond that of young mice. Finally, we identified the transcription factor Ebf3 as a downstream mediator of Notch signaling in SSPCs that is dysregulated with aging, highlighting it as a promising therapeutic target to rejuvenate the aged skeleton.

Bone Tissue Engineering (BTE) of the Craniofacial Skeleton, Part I: Evolution and Optimization of 3D-Printed Scaffolds for Repair of Defects.

Bone tissue regeneration is a complex process that proceeds along the well-established wound healing pathway of hemostasis, inflammation, proliferation, and remodeling. Recently, tissue engineering efforts have focused on the application of biological and technological principles for the development of soft and hard tissue substitutes. Aim is directed towards boosting pathways of the healing process to restore form and function of tissue deficits. Continued development of synthetic scaffolds, cell therapies, and signaling biomolecules seeks to minimize the need for autografting. Despite being the current gold standard treatment, it is limited by donor sites' size and shape, as well as donor site morbidity. Since the advent of computer-aided design/computer-aided manufacturing (CAD/CAM) and additive manufacturing (AM) techniques (3D printing), bioengineering has expanded markedly while continuing to present innovative approaches to oral and craniofacial skeletal reconstruction. Prime examples include customizable, high-strength, load bearing, bioactive ceramic scaffolds. Porous macro- and micro-architecture along with the surface topography of 3D printed scaffolds favors osteoconduction and vascular in-growth, as well as the incorporation of stem and/or other osteoprogenitor cells and growth factors. This includes platelet concentrates (PCs), bone morphogenetic proteins (BMPs), and some pharmacological agents, such as dipyridamole (DIPY), an adenosine A 2A receptor indirect agonist that enhances osteogenic and osteoinductive capacity, thus improving bone formation. This two-part review commences by presenting current biological and engineering principles of bone regeneration utilized to produce 3D-printed ceramic scaffolds with the goal to create a viable alternative to autografts for craniofacial skeleton reconstruction. Part II comprehensively examines recent preclinical data to elucidate the potential clinical translation of such 3D-printed ceramic scaffolds.

Osteogenic differentiation and reconstruction of mandible defects using a novel resorbable membrane: An in vitro and in vivo experimental study.

To evaluate the cellular response of both an intact fish skin membrane and a porcine-derived collagen membrane and investigate the bone healing response of these membranes using a translational, preclinical, guided-bone regeneration (GBR) canine model. Two different naturally sourced membranes were evaluated in this study: (i) an intact fish skin membrane (Kerecis Oral®, Kerecis) and (ii) a porcine derived collagen (Mucograft®, Geistlich) membrane, positive control. For the in vitro experiments, human osteoprogenitor (hOP) cells were used to assess the cellular viability and proliferation at 24, 48, 72, and 168 h. ALPL, COL1A1, BMP2, and RUNX2 expression levels were analyzed by real-time PCR at 7 and 14 days. The preclinical component was designed to mimic a GBR model in canines (n = 12). The first step was the extraction of premolars (P1-P4) and the 1st molars bilaterally, thereby creating four three-wall box type defects per mandible (two per side). Each defect site was filled with bone grafting material, which was then covered with one of the two membranes (Kerecis Oral® or Mucograft®). The groups were nested within the mandibles of each subject and membranes randomly allocated among the defects to minimize potential site bias. Samples were harvested at 30-, 60-, and 90-days and subjected to computerized microtomography (µCT) for three-dimensional reconstruction to quantify bone formation and graft degradation, in addition to histological processing to qualitatively analyze bone regeneration. Neither the intact fish skin membrane nor porcine-based collagen membrane presented cytotoxic effects. An increase in cell proliferation rate was observed for both membranes, with the Kerecis Oral® outperforming the Mucograft® at the 48- and 168-hour time points. Kerecis Oral® yielded higher ALPL expression relative to Mucograft® at both 7- and 14-day points. Additionally, higher COL1A1 expression was observed for the Kerecis Oral® membrane after 7 days but no differences were detected at 14 days. The membranes yielded similar BMP2 and RUNX2 expression at 7 and 14 days. Volumetric reconstructions and histologic micrographs indicated gradual bone ingrowth along with the presence of particulate bone grafts bridging the defect walls for both Kerecis Oral® and Mucograft® membranes, which allowed for the reestablishment of the mandible shape after 90 days. New bone formation significantly increased from 30 to 60 days, and from 60 to 90 days in vivo, without significant differences between membranes. The amount of bovine grafting material (%) within the defects significantly decreased from 30 to 90 days. Collagen membranes led to an upregulation of cellular proliferation and adhesion along with increased expression of genes associated with bone healing, particularly the intact fish skin membrane. Despite an increase in the bone formation rate in the defect over time, there was no significant difference between the membranes.

Employing Indirect Adenosine 2A Receptors (A2AR) to Enhance Osseointegration of Titanium Devices: A Pre-Clinical Study.

The present study aimed to evaluate the effect of dipyridamole, an indirect adenosine 2A receptors (A2AR), on the osseointegration of titanium implants in a large, translational pre-clinical model. Sixty tapered, acid-etched titanium implants, treated with four different coatings ((i) Type I Bovine Collagen (control), (ii) 10 µM dipyridamole (DIPY), (iii) 100 µM DIPY, and (iv) 1000 µM DIPY), were inserted in the vertebral bodies of 15 female sheep (weight ~65 kg). Qualitative and quantitative analysis were performed after 3, 6, and 12 weeks in vivo to assess histological features, and percentages of bone-to-implant contact (%BIC) and bone area fraction occupancy (%BAFO). Data was analyzed using a general linear mixed model analysis with time in vivo and coating as fixed factors. Histomorphometric analysis after 3 weeks in vivo revealed higher BIC for DIPY coated implant groups (10 µM (30.42% ± 10.62), 100 µM (36.41% ± 10.62), and 1000 µM (32.46% ± 10.62)) in comparison to the control group (17.99% ± 5.82). Further, significantly higher BAFO was observed for implants augmented with 1000 µM of DIPY (43.84% ± 9.97) compared to the control group (31.89% ± 5.46). At 6 and 12 weeks, no significant differences were observed among groups. Histological analysis evidenced similar osseointegration features and an intramembranous-type healing pattern for all groups. Qualitative observation corroborated the increased presence of woven bone formation in intimate contact with the surface of the implant and within the threads at 3 weeks with increased concentrations of DIPY. Coating the implant surface with dipyridamole yielded a favorable effect with regard to BIC and BAFO at 3 weeks in vivo. These findings suggest a positive effect of DIPY on the early stages of osseointegration.

Patient-specific 3D printed Poly-ether-ether-ketone (PEEK) dental implant system.

Fused Filament Fabrication (FFF)-based 3D printing is an efficient technique for developing medical implants, but it is not very useful in developing small yet mechanically robust design-specific fixtures such as dental implants (<15 mm). Specifically, it is challenging to 3D print robust Polyetheretherketone (PEEK) small implants due to PEEK's high melting temperature and melt viscosity. However, in this study, we efficiently utilize high-temperature FFF to develop the first-of-its-kind patient-specific robust PEEK dental implants with high print resolution. Specifically, we explore the effects of critical FFF processing conditions on the mechanical properties of the implants and subsequently determine an optimized set of processing conditions that are essential in developing durable dental implant systems. Our results indicate that the 3D printed dental implants exhibit good fatigue properties and suffice the clinical and industrial requirements for dental implants. Furthermore, we prove that the 3D printed implants exhibit adequate mechanical durability even after simulated (accelerated) aging of 30 years.