Regeneration Of Critical-sized Defects Research Articles

Regeneration of Critical-Sized Mandibular Defects Using 3D-Printed Composite Scaffolds: A Quantitative Evaluation of New Bone Formation in In Vivo Studies.

Mandibular tissue engineering aims to develop synthetic substitutes for the regeneration of critical size defects (CSD) caused by a variety of events, including tumor surgery and post-traumatic resections. Currently, the gold standard clinical treatment of mandibular resections (i.e., autologous fibular flap) has many drawbacks, driving research efforts toward scaffold design and fabrication by additive manufacturing (AM) techniques. Once implanted, the scaffold acts as a support for native tissue and facilitates processes that contribute to its regeneration, such as cells infiltration, matrix deposition and angiogenesis. However, to fulfil these functions, scaffolds must provide bioactivity by mimicking natural properties of the mandible in terms of structure, composition and mechanical behavior. This review aims to present the state of the art of scaffolds made with AM techniques that are specifically employed in mandibular tissue engineering applications. Biomaterials chemical composition and scaffold structural properties are deeply discussed, along with strategies to promote osteogenesis (i.e., delivery of biomolecules, incorporation of stem cells, and approaches to induce vascularization in the constructs). Finally, a comparison of in vivo studies is made by taking into consideration the amount of new bone formation (NB), the CSD dimensions, and the animal model.

Bone Tissue Engineering Using Osteogenic Cells: From the Bench to the Clinical Application.

The use of tissue engineering to restore and to build new bone tissue is under active research at present. The following review summarizes the latest studies and clinical trials related to the use of osteogenic cells, biomaterials, and scaffolds to regenerate bone defects in the human jaws. Bone tissue engineering (BTE) combined with scaffolds have provided a range of advantages not only to transport the target cells to their desired destination but also to support the early phases of the mineralization process. The mechanical, chemical, and physical properties of scaffolds have been evaluated as they affect the quantity of bone regeneration, particularly in the oral cavity. This review also highlighted the mechanisms underlying bone homeostasis, including the key transcription factors and signaling pathways responsible for regulating the differentiation of osteoblast lineage. Furthering understanding of the mechanisms of cellular signaling in skeletal remodeling with the use of mesenchymal stem cells and the proper scaffold properties are key-factors to enable the incorporation of new and effective treatment methods into clinical practice for bone tissue regeneration using BTE. Impact Statement The use of mesenchymal stem cells able to differentiate in osteoblast lineage for bone tissue engineering (BTE) remains a major challenge. Viable cells and signaling pathways play an essential role in bone repair and regeneration of critical size defects. Recent advances in scaffolds and biological factors such as growth factors (e.g., cytokines and hormones) controlling the osteogenic signaling cascade are now becoming new players affecting the osteogenic potential of cells. Such techniques will significantly impact the maxillofacial bone tissue replacement, repair, and regeneration for patients without having to rely on donor banks or other surgical sites.

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 regeneration 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 (14 mm) for 3, 6 and 12 weeks. The results showed that HG improved the handling properties of the Bonelike® granules and did not affect its osteoconductive features, neither impairing the bone regeneration 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.

Effect of silicon-doped calcium phosphate bone grafting materials on bone regeneration and osteogenic marker expression after implantation in the ovine scapula.

Compared to the currently clinically available bone grafting materials for alveolar ridge augmentation, there is a great demand for bioactive bone substitutes with higher resorbability, which enhance osteogenesis at the same time. This has prompted the development of a silicon-doped rapidly resorbable calcium alkali orthophosphate (Si-CAOP) and silicon-doped β-tricalcium phosphate (Si-TCP). This study evaluated the effect of these two particulate graft materials as compared to the currently clinically used β-TCP on bone formation and osteogenic marker expression after 2 weeks, 1, 3, 6, 12, and 18 months of implantation in critical size defects in the sheep scapula. Immunohistochemical analysis of collagen type I, alkaline phosphatase, and osteocalcin expression was performed on resin embedded sections. The bone and particle area fraction and the bone-biomaterial contact were determined histomorphometrically. After 2 weeks and 1 month defects grafted with Si-CAOP displayed a significantly greater bone area fraction, bone-particle-contact, osteogenic marker expression and significantly lower particle area fraction than defects grafted with Si-TCP and TCP. By 3 and 6 months all materials studied mediated excellent defect regeneration with further bone remodeling at 12 and 18 months. Taken together, Si-CAOP induced the most expeditious bone regeneration of critical size defects in the sheep scapula. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 2018. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater 107B: 594-614, 2019.

Cell therapy with allogenic canine periodontal ligament-derived cells in periodontal regeneration of critical size defects.

The objective of this in vivo experimental study to evaluate the regenerative potential of a cell therapy combining allogenic periodontal ligament-derived cells within a xenogeneic bone substitute in a similar experimental model. In nine beagle dogs, critical size 6-mm supra-alveolar periodontal defects were created around the PIII and PIV. The resulting supra-alveolar defects were randomly treated with either 1.4×106 allogenic canine periodontal ligament-derived cells seeded on de-proteinized bovine bone mineral with 10% collagen (DBBM-C) (test group) or DBBM-C without cells (control group). Specimens were obtained at 3months, and histological outcomes were studied. The histological analysis showed that total furcation closure occurred very seldom in both groups, being the extent of periodontal regeneration located in the apical third of the defect. The calculated amount of periodontal regeneration at the furcation area was comparable in both the test and control groups (1.93±1.14mm (17%) versus 2.35±1.74mm (22%), respectively (p=.37). Similarly, there were no significant differences in the amount of new cementum formation 4.49±1.56mm (41%) versus 4.97±1.05mm (47%), respectively (p=.45). This experimental study was unable to demonstrate the added value of allogenic cell therapy in supra-crestal periodontal regeneration.

Promoting Cell Migration in Tissue Engineering Scaffolds with Graded Channels.

Ideal bone scaffolds having good biocompatibility, good biodegradability, and beneficial mechanical properties are the basis for bone tissue engineering. Specifically, cell migration within 3D scaffolds is crucial for bone regeneration of critical size defects. In this research, hydroxyapatite scaffolds with three different types of architectures (tortuous, parallel, and graded channels) are fabricated using the freeze-casting (ice-templating) method. While most studies promote cell migration by chemical factors, it can be greatly enhanced by introducing only graded channels as compared with tortuous or parallel channels. The results provide insights and guidance in designing novel scaffolds to enhance cell migration behavior for bone tissue regeneration.

Vascularization Potential of Electrospun Poly(L-Lactide-co-Caprolactone) Scaffold: The Impact for Tissue Engineering.

BackgroundElectrospun nanofibers have widespread putative applications in the field of regenerative medicine and tissue engineering. When compared to naturally occurring collagen matrices, electrospun nanofiber scaffolds have two distinct advantages: they do not induce a foreign body reaction and they are not at risk for biological contamination. However, the exact substrate, structure, and production methods have yet to be defined.Material/MethodsIn the current study, tubular-shaped poly(L-lactide-co-caprolactone) (PLCL) constructs produced using electrospinning technology were evaluated for their potential application in the field of tissue regeneration in two separate anatomic locations: the skin and the abdomen. The constructs were designed to have an internal diameter of 3 mm and thickness of 200 μm. Using a rodent model, 20 PLCL tubular constructs were surgically implanted in the abdominal cavity and subcutaneously. The constructs were then evaluated histologically using electron microscopy at 6 weeks post-implantation.ResultsHistological evaluation and analysis using scanning electron microscopy showed that pure scaffolds by themselves were able to induce angiogenesis after implantation in the rat model. Vascularization was observed in both tested groups; however, better results were obtained after intraperitoneal implantation. Formation of more and larger vessels that migrated inside the scaffold was observed after implantation into the peritoneum. In this group no evidence of inflammation and better integration of scaffold with host tissue were noticed. Subcutaneous implantation resulted in more fibrotic reaction, and differences in cell morphology were also observed between the two tested groups.ConclusionsThis study provides a standardized evaluation of a PLCL conduit structure in two different anatomic locations, demonstrating the excellent ability of the structure to achieve vascularization. Functional, histological, and mechanical data clearly indicate prospective clinical utilization of PLCL in critical size defect regeneration.

Periosteum tissue engineering in an orthotopic invivo platform.

The periosteum plays a critical role in bone homeostasis and regeneration. It contains a vascular component that provides vital blood supply to the cortical bone and an osteogenic niche that acts as a source of bone-forming cells. Periosteal grafts have shown promise in the regeneration of critical size defects, however their limited availability restricts their widespread clinical application. Only a small number of tissue-engineered periosteum constructs (TEPCs) have been reported in the literature. A current challenge in the development of appropriate TEPCs is a lack of pre-clinical models in which they can reliably be evaluated. In this study, we present a novel periosteum tissue engineering concept utilizing a multiphasic scaffold design in combination with different human cell types for periosteal regeneration in an orthotopic invivo platform. Human endothelial and bone marrow mesenchymal stem cells (BM-MSCs) were used to mirror both the vascular and osteogenic niche respectively. Immunohistochemistry showed that the BM-MSCs maintained their undifferentiated phenotype. The human endothelial cells developed into mature vessels and connected to host vasculature. The addition of an invitro engineered endothelial network increased vascularization in comparison to cell-free constructs. Altogether, the results showed that the human TEPC (hTEPC) successfully recapitulated the osteogenic and vascular niche of native periosteum, and that the presented orthotopic xenograft model provides a suitable invivo environment for evaluating scaffold-based tissue engineering concepts exploiting human cells.

Skull bone regeneration through the application of tissue engineering techniques

Skull bone regeneration through the application of tissue engineering techniques

Microdialysis in critical size bone defects � quantitative assessment of cytokines and proteins in the early stage of fracture repair

Event Abstract Back to Event Microdialysis in critical size bone defects – quantitative assessment of cytokines and proteins in the early stage of fracture repair Yvonne Förster1, Johannes Schmidt2, Dirk K. Wissenbach3, Susanne E. Pfeiffer3, Martin Von Bergen2, 3, Stefan Kalkhof2 and Stefan Rammelt1 1 University Hospital "Carl Gustav Carus" at TU Dresden, UniversityCenter of Orthopedics and Trauma Surgery, Germany 2 Helmholtz-Centre for Environmental Research-UFZ, Department of Proteomics, Germany 3 Helmholtz-Centre for Environmental Research-UFZ, Department of Metabolomics, Germany Fracture repair is a highly complex process influenced by growth factors, cytokines and other mediators. The early stages of the bone healing are of specific interest as they are essential for later consolidation but still poorly understood. From model systems we know about the significance of single proteins, but knowledge about the processes directly within the bone defect is limited. This is also caused by a lack of methods that allow the sampling of growth factors, cytokines and metabolites in situ. Microdialysis is a promising approach to obtain time-resolved data of specific growth factors and cytokines directly from the fracture site in vivo. When combined with a comprehensive analysis of the proteins adsorbed at the catheter tip, complementary data can be gathered from the wound site of the same defect. A 5 mm defect was created in the femur of 12 adult male Wistar rats and stabilized with an internal fixator. The microdialysis catheter was placed within the defect and the wound fluid was collected continuously under anesthesia for 24 h. Twelve rats with only a soft tissue wounds without bone defects were included as controls. Samples were collected in 3 h intervals and used to quantify cytokines and growth factors by ELISA. At the end of experiment the microdialysis catheters were explanted, the proteins adsorbed to the membrane of the microdialysis catheter were extracted and were analyzed after separation by HPLC-MS/MS. The functional annotation clustering was performed using the software packages PANTHER and DAVID.[1],[2] Analysis of the wound fluid by ELISA resulted in concentration profiles for 9 different cytokines and growth factors. Detailed analysis of Interleukin-6 (IL-6) showed that the concentration rapidly increased after surgery and declined to baseline after 24 h (Fig. 1A). In contrast, the concentration of the potentially relevant chemokine CXCL7 ((C-X-C) motif ligand 7) showed a rapid decrease immediately after surgery suggesting the release of the protein from activated platelets (Fig. 1B). More than 500 proteins were identified at the membrane of the catheter. Among them 222 proteins were detected in at least 3 of 4 replicates in both groups indicating a good reliability of the method. We could detect 78 proteins that occurred either exclusively (31) or with significantly increased abundance (47) within bone defect. For the control group, only 11 proteins were exclusive or significant increased compared to the group with a bone defect (Fig. 2). The combination of microdialysis with proteomics and metabolomics is a promising approach to obtain a detailed and direct insight into the local extracellular microenvironment of a bone defect. With these methods it is possible to obtain comprehensive and time-resolved information from the early stages of fracture repair. These data are of potential use for the development of new biomaterials that are designed to enhance bone repair and regeneration of critical size defects. Fig 1: A) Protein concentration of IL-6 in the wound fluid from bone defect (n=6) and soft tissue wound (n=8). B) Protein concentration of CXCL7 in the wound fluid from bone defect (n= 4) and soft tissue wound (n=4). Fig. 2: Distribution of the identified proteins at the membrane of the catheter. For each defect scenario 4 catheters were analyzed. This study was supported by grants from Deutsche Forschungsgemeinschaft Transregio 67 (subprojects B1, B5, and Z4). The authors thank Ute Hempel, Carolin Preissler, Sven Baumann, and Jacqueline Kober for technical assistance.

Effect of Silicon-Doped Calcium Phosphate Bone Substitutes on Bone Formation and Osteoblastic Phenotype Expression <i>In Vivo</i>

This study evaluates the effect of two novel particulate silicon-doped calcium phosphate graft materials as compared to the currently clinically used material β-TCP on osteogenesis and bone formation after implantation in critical-size defects the sheep scapula. These materials were developed in order to create biodegradable bone substitute materials that degrade rapidly, but still stimulate osteogenesis at the same time, thereby resulting in bone repair and regeneration with fully functional bone tissue. All bone substitute materials studied facilitated excellent bony regeneration of critical-size defects in the sheep scapula. Of the three grafting materials studied, the calcium alkali orthophosphate material with the crystalline phase Ca2KNa (PO4)2, with a small amorphous portion containing magnesium potassium phosphate and a small addition of sodium magnesium silicate had the greatest stimulatory effect on bone formation and expression of osteogenic markers, while exhibiting the highest biodegradability.

Critical size defect regeneration by rhPTH-collagen membrane as a new tissue engineering tool

Recombinant Human Parathyroid Hormone (rhPTH 1-34) administration is an effective treatment to improve bone mass in osteoporosis. The aim of this study was to develop a Tissue Engeenering Tool for bone regeneration. We evaluated the efficacy of a freeze dried rhPTH membrane in calvarial critical size defect (CSD). Forty-four Wistar female rats (body weight 150 ± 50 g) with CSD (5 mm) were divided into four groups: group 1: rhPTH membrane (rhPTHm); group 2: atelocollagen membrane (Cm); group 3: rhPTH and atelocollagen I (CrhPTHm); group 4: without any treatment (CG). All samples were evaluated on the 1st, 3rd, and 6th weeks (weeks) post-surgery by soft X-ray, histological and histometric studies. Soft X-ray results showed a radiolucent image with many irregular radiopaque areas. Histologically, rhPTHm was replaced by reticular bone (7%) since 3rd week, and lamellar bone ossicles (30%) at 6th week. Cm showed bone formation like composite bone type on week 1st, 3rd, and 6th (2%, 44%, and 41%, respectively). With CrhPTHm, bone formation was observed in all periods (2.4%, 48%, and 53%), showing statistical difference with CG in the 3rd and 6th wks (p = 0.03 and 0.01). Our results demonstrated the effectiveness of a new biomaterial called CrhPTHm because its ability to regenerate calvarial CSD. Moreover, the membrane represents a new local intermittent delivery system allowing rhPTH slow release.

Critical size defect regeneration using PEG-mediated BMP-2 gene delivery and the use of cell occlusive barrier membranes - the osteopromotive principle revisited

The objective of this study was to investigate if osseous regeneration can be accelerated by involvement of periosteal tissue. Bone defect regeneration could be accelerated by the involvement of periosteal tissue if osteogenic cell signalling is maintained within the defect. It was questioned if local cell-mediated BMP-2 gene delivery makes a cell occlusive membrane dispensable during bone critical size defect regeneration. PEG matrix (degradation time 10days) and PEG membrane (degradation time 120days) were used in the pig calvarial model. Cylindrical (1×1cm) critical size defects (CSD) (9 per animal; 20 animals) were filled with: (i) particulated autologous bone, covered with PEG membrane (group 1); (ii) HA/TCP, covered with PEG membrane (group 2); (iii) HA/TCP, mixed with PEG matrix (group 3); and (iv) HA/TCP mixed with BMP-2-transfected osteoblasts and PEG matrix (group 4). BMP-2/4 gene transfer: liposomal in vitro transfection of BMP-2/V5-tag fusion-protein. Quantitative histomorphometry (toluidine blue staining) after 2, 4 and 12weeks assessed bone formation. Semiquantitative immunohistochemistry estimated the expression of BMP-2, V5-tag, Runx-2 and Sox9. PEG matrix embedded BMP-2 expressing cells presented higher bone formation (P<0.05) than HA/TCP+PEG matrix defect filling or PEG membrane covering (HA/TCP filling) after 12weeks. Highest expression of BMP-2, Runx-2 and lowest expression of fibrous tissue marker Sox9 was seen in the BMP-2 group. PEG matrix embedded BMP-2 expressing cells are capable to maintain osteogenic signalling and to accelerate osseous defect regeneration in absence of a cell occlusive membrane.

PEG matrix enables cell-mediated local BMP-2 gene delivery and increased bone formation in a porcine critical size defect model of craniofacial bone regeneration

This study addressed the suitability of a polyethylene glycol (PEG) matrix as scaffold for cell-mediated local BMP-2 gene transfer in a calvarial critical size defect (CSD) model. PEG matrix (degradation time 10 days) and PEG membrane (degradation time 120 days) were used in the pig calvarial model. Cylindrical (1 × 1 cm) CSD (9 per animal; 20 animals) were filled with: (i) HA/TCP, covered by PEG membrane (group 1); (ii) HA/TCP, mixed with PEG matrix (group 2); and (iii) HA/TCP mixed with BMP-2 transfected osteoblasts and PEG matrix (group 3). BMP-2/4 gene transfer: liposomal in vitro transfection of BMP-2/V5-tag fusion-protein. Quantitative histomorphometry (toluidine blue staining) after 2, 4 and 12 weeks assessed bone formation. Semiquantitative immunohistochemistry estimated the expression of BMP-2 and V5-tag. Group 3 showed significantly higher new bone formation than groups 1, 2 at 4 (P < 0.05) and 12 (P < 0.02) weeks. BMP-2-V5-tag was detected for 4 weeks. BMP-2 expression in group 3 was higher compared to all other groups after 2 and 4 (P < 0.02) weeks. The PEG matrix serves as scaffold for cell-mediated BMP-2 gene delivery in guided bone regeneration facilitating cell survival and protein synthesis for at least 4 weeks. Local BMP-2 gene delivery by PEG matrix-embedded cells leads to increased bone formation during critical size defect regeneration.

Xenopus Laevisas a Novel Model to Study Long Bone Critical-Size Defect Repair by Growth Factor-Mediated Regeneration

We used the tarsus of an adult Xenopus laevis frog as an in vivo load-bearing model to study the regeneration of critical-size defects (CSD) in long bones. We found the CSD for this bone to be about 35% of the tarsus length. To promote regeneration, we implanted biocompatible 1,6 hexanediol diacrylate scaffolds soaked with bone morphogenetic proteins-4 and vascular endothelial growth factors. In contrast to studies that use scaffolds as templates for bone formation, we used scaffolds as a growth factor delivery vehicle to promote cartilage-to-bone regeneration. Defects in control frogs were filled with scaffolds lacking growth factors. The limbs were harvested at a series of time points ranging from 3 weeks to 6 months after implantation and evaluated using micro-computed tomography and histology. In frogs treated with growth factor-loaded scaffolds, we observed a cartilage-to-bone regeneration in the skeletal defect. Five out of eight defects were completely filled with cartilage by 6 weeks. Blood vessels had invaded the cartilage, and bone was beginning to form in ossifying centers. By 3 months, these processes were well advanced, and extensive ossification was observed in 6-month samples. In contrast, the defects in control frogs showed only formation of fibrous scar tissue. This study demonstrates the utility of a Xenopus model system for tissue engineering research and that the normal in vivo mechanism of endochondral bone development and fracture repair can be mimicked in the repair of CSD with scaffolds used as growth factor delivery mechanisms.