Critical Calvarial Defects Research Articles

Antler Collagen/Chitosan Scaffolds Improve Critical Calvarial Defect Healing in Rats

Antler Collagen/Chitosan Scaffolds Improve Critical Calvarial Defect Healing in Rats

Evaluating the bone regeneration in calvarial defect using osteoblasts differentiated from adipose-derived mesenchymal stem cells on three different scaffolds: an animal study.

The aim of this study was to investigate the effect of three different scaffolds on the viability and differentiation of adipose-derived mesenchymal stem cells (ADMSCs) to osteoblast for bone regeneration of calvarial defect in rabbit model. Adipose was harvested from the nape of 12 rabbits by direct surgery or hollow-tip cannula. Two standardized circular calvarial defects (case and control), 8 mm in diameter each, were created in all the animals. The animals were divided into 3 different groups. In group 1 (G1), the defect was filled with polyamide + ADMSC. In group 2, poly lactic-co-glycolic acid + ADMSC was used. In group 3, decellularized amniotic membrane + ADMSC was applied. In the control defect, the non-seeded scaffolds were applied for filling the defect. Decellularized pericardial scaffolds were used as a membrane on the scaffolds. The animals were euthanized 2, 4, and 8 weeks of operation and new bone formation was assessed by different analyses. Immunohistochemical (IHC) staining with osteopontin and osteocalcin antibodies was also performed. After 2 weeks of wound healing, minimal bone regeneration was detected in all groups. Almost complete defect closure was observed in all experimental groups after 8 weeks of operation, with the greatest defect closure in the animals treated with polyamide scaffolds as compared to biopsies obtained from control defects and other experimental groups. The maximal tensile load was higher in G1, 4 and 8 weeks postoperatively, suggesting the usefulness of polyamide + ADMSC for bone regeneration in calvarial defects. Results of the IHC staining demonstrated a significant difference between seeded and non-seeded scaffold in both short- and long-term follow-ups (P < 0.05). In addition, a significant difference was observed in enhancement of IHC staining of both markers in polyamide group (seeded or non-seeded) 4 and 8 weeks postoperatively in comparison with other scaffolds. It was concluded that bone regeneration in critical calvarial defect was more successful in seeded polyamide.

Osseointegrative properties of electrospun hydroxyapatite-containing nanofibrous chitosan scaffolds.

Our long-term goal is to develop smart biomaterials that can facilitate regeneration of critical-size craniofacial lesions. In this study, we tested the hypothesis that biomimetic scaffolds electrospun from chitosan (CTS) will promote tissue repair and regeneration in a critical size calvarial defect. To test this hypothesis, we first compared in vitro ability of electrospun CTS scaffolds crosslinked with genipin (CTS-GP) to those of mineralized CTS-GP scaffolds containing hydroxyapatite (CTS-HA-GP), by assessing proliferation/metabolic activity and alkaline phosphatase (ALP) levels of murine mesenchymal stem cells (mMSCs). The cells' metabolic activity exhibited a biphasic behavior, indicative of initial proliferation followed by subsequent differentiation for all scaffolds. ALP activity of mMSCs, a surrogate measure of osteogenic differentiation, increased over time in culture. After 3 weeks in maintenance medium, ALP activity of mMSCs seeded onto CTS-HA-GP scaffolds was approximately two times higher than that of cells cultured on CTS-GP scaffolds. The mineralized CTS-HA-GP scaffolds were also osseointegrative in vivo, as inferred from the enhanced bone regeneration in a murine model of critical size calvarial defects. Tissue regeneration was evaluated over a 3 month period by microCT and histology (Hematoxylin and Eosin and Masson's Trichrome). Treatment of the lesions with CTS-HA-GP scaffolds induced a 38% increase in the area of de novo generated mineralized tissue area after 3 months, whereas CTS-GP scaffolds only led to a 10% increase. Preseeding with mMSCs significantly enhanced the regenerative capacity of CTS-GP scaffolds (by ∼3-fold), to 35% increase in mineralized tissue area after 3 months. CTS-HA-GP scaffolds preseeded with mMSCs yielded 45% new mineralized tissue formation in the defects. We conclude that the presence of HA in the CTS-GP scaffolds significantly enhances their osseointegrative capacity and that mineralized chitosan-based scaffolds crosslinked with genipin may represent a unique biomaterial with possible clinical relevance for the repair of critical calvarial bone defects.

Micro‐CT and PET analysis of bone regeneration induced by biodegradable scaffolds as carriers for dental pulp stem cells in a rat model of calvarial “critical size” defect: Preliminary data

Bone regeneration strategies in dentistry utilize biodegradable scaffolds seeded with stem cells able to induce bone formation. However, data on regeneration capacity of these tissue engineering constructs are still deficient. In this study micro-Computed tomography (micro-CT) and positron emission tomography (PET) analyses were used to investigate bone regeneration induced by two scaffolds [Granular deproteinized bovine bone (GDPB) and Beta-tricalcium phosphate (β-TCP)] used alone or in combination with dental pulp stem cells (DPSC) in a tissue engineered construct implanted in a rat critical calvarial defect. Bone mineral density (BMD) and standard uptake value (SUV) of tracer incorporation were measured after 2, 4, 8, and 12 weeks post-implant. The results showed that: (1) GDPB implants were mostly well positioned, as compared to ß-TCP; (2) GDPB induced higher BMD and SUV values within the cranial defect as compared to ß-TCP, either alone or in combination with stem cells; (3) addition of DPSC to the grafts did not significantly induce an increase in BMD and SUV values as compared to the scaffolds grafted alone, although a small tendency to increase was observed. Thus our study demonstrates that GDPB, when used to fill critical calvarial defects, induces a greater percentage of bone formation as compared to ß-TCP. Moreover, this study shows that addition of DPSC to pre-wetted scaffolds has the potential to ameliorate bone regeneration process, although the set of optimal conditions requires further investigation.

A Comparative Morphometric Analysis of Biodegradable Scaffolds as Carriers for Dental Pulp and Periosteal Stem Cells in a Model of Bone Regeneration

Bone regeneration and bone fixation strategies in dentistry utilize scaffolds containing regenerating-competent cells as a replacement of the missing bone portions and gradually replaced by autologous tissues. Mesenchymal stem cells represent an ideal cell population for scaffold-based tissue engineering. Among them, dental pulp stem cells (DPSCs) and periosteal stem cells (PeSCs) have the potential to differentiate into a variety of cell types including osteocytes, suggesting that they can be used with this purpose. However, data on bone regeneration properties of these types of cells in scaffold-based tissue engineering are yet insufficient.In this study, we evaluated temporal dynamic bone regeneration (measured as a percentage of bone volume on the total area of the defect) induced by DPSCs or PeSCs when seeded with different scaffolds to fill critical calvarial defects in SCID Beige nude mice. Two commercially available scaffolds (granular deproteinized bovine bone with 10% porcine collagen and granular β;-tricalcium phosphate) and one not yet introduced on the market (a sponge of agarose and nanohydroxyapatite) were used. The results showed that tissue-engineered constructs did not significantly improve bone-induced regeneration process when compared with the effect of scaffolds alone. In addition, the data also showed that the regeneration induced by β;-tricalcium phosphate alone was higher after 8 weeks than that of scaffold seeded with the 2 stem cell lines. Altogether these findings suggest that further studies are needed to evaluate the potential of DPSCs and PeSCs in tissue construct and identify the appropriate conditions to generate bone tissue in critical-size defects.

Effect of Adipose Tissue-Derived Osteogenic and Endothelial Cells on Bone Allograft Osteogenesis and Vascularization in Critical-Sized Calvarial Defects

The use of processed bone allograft to repair large osseous defects of the skull has been limited, given that it lacks the osteogenic cellularity and intrinsic vascular supply which are essential elements for successful graft healing and, at the same time, the areas to be targeted through tissue-engineering applications. In this study, we investigated the effect of predifferentiated rat adipose tissue-derived osteoblastic cells (OBs) and endothelial cells (ECs) on calvarial bone allograft healing and vascularization using an orthotopic critical-sized calvarial defect model. For this purpose, thirty-seven 8 mm critical calvarial defects in Lewis rats were treated with bone allografts seeded with no cells, undifferentiated adipose tissue-derived stem cells (ASC), OBs, ECs, and OBs and ECs simultaneously. After 8 weeks, the bone volume and mineral density were calculated using microcomputed tomography and the microvessel formation using immunohistochemical staining and imaging software. The amount of bone within the 8 mm defect was significantly higher for the allografts treated with ECs compared with the allografts treated with OBs (p=0.05) and simultaneously with the two cell lineages (p=0.02). There were no significant differences in bone formation between the latter two groups and the control groups (allografts treated with no cells and undifferentiated ASC). There were no significant differences in bone mineral density among the groups. The amount of microvessels was significantly higher in the group treated with ECs relative to all groups (p=< 0.05). Our results show that the implantation of ASC-derived ECs improves the vascularization of calvarial bone allografts at 8 weeks after treatment. This cell-based vascularization strategy can be used to improve the paucity of perfusion in allogenic bone implants. However, in this study, the treatment of allografts with OBs alone or in combination with ECs did not support bone formation or vascularization.

Association of mesenchymal stem cells with platelet rich plasma on the repair of critical calvarial defects in mice

To evaluate the effects of mesenchymal stem cells (MSC) from eight mice C57BL/6 gfp(+) bone marrows expanded in cultures associated with platelets rich plasma (PRP) deriving from another eight mice, in the repair of critical defects in calvarial bone produced in twenty-four adult isogenic mice C57BL/6. The animals were submitted to a cranial defect of 6.0mm in diameter and divided into two equal experimental groups. Control group did not receive treatment and the treated group received a MSC pellet containing 1.0 x 10(7) cells/mL associated with 50.0 µL of plasma gel containing 1.0 x 10(9) autologous platelets within the defect. In the treated group was observed process of angiogenesis and bone repair better than control group. Mesenchymal stem cells derived from bone marrow of C57BL/6 gfp(+) mice associated with PRP gel applied in bone critical defects produced in calvarial contributes positively to the process of bone repair.

Osteogenic Induction of Adipose‐derived Stromal Cells: Not a Requirement for Bone Formation In Vivo

Osteogenic induction was regarded as an indispensable step for adipose-derived stromal cells (ADSCs) to have osteogenic ability. Non-induced ADSCs can also produce bone in vivo and heal skeletal defects. The present study aimed to compare the bone-forming ability of osteogenically induced ADSCs and non-induced ADSCs in vivo. Tissue-engineered constructs were prepared from osteogenically induced or non-induced ADSCs and porous hydroxyapatite/beta-tricalcium phosphate scaffolds. A scaffold without cells and an empty defect group were used as control. All were implanted in rat critical calvarial defects. After implantation for 6 and 12 weeks, bone formation was analyzed using histomorphometry and microcomputed tomography; there were no significant differences in the formation of new bone between osteogenically induced ADSCs and non-induced ADSCs (P > 0.05). In conclusion, osteogenic induction of ADSCs is not an indispensable step for bone formation in vivo. Non-induced ADSCs can also be used as seeding cells to construct bone tissue.

Role of Platelet Gel and Calcium-Coated Lactosorb Membranes in Healing Critical Calvarial Defects

We evaluated the efficacy of coating Lactosorb (Biomet Inc, Warsaw, IN) membranes with calcium hydroxyapatite in critical-size defects in rabbits. We also evaluated whether platelet gel affects calvarial bone healing.Fifteen adult New Zealand white rabbits were divided into 5 groups. Each animal underwent a surgical procedure in which 2 critical-size (15 mm) calvarial defects were created in the parietal bones. Group A was control. Group B was a control in 1 defect and Lactosorb in the other defect. Group C was treated with Lactosorb in 1 defect and Lactosorb coated with calcium hydroxyapatite in the other defect. Group D was treated with Lactosorb in 1 defect and coated Lactosorb without calcium hydroxyapatite in the other defect. Group E was treated with platelet gel in 1 defect and control in the other. The animals were killed at 1, 3, and 6 months. The bones were excised and evaluated by clinical observation, radiography, and histology.None of the defects were closed at 6 months. Defects treated with platelet gel healed in a similar fashion as control. All defects treated with a barrier (Lactosorb, Lactosorb coated, Lactosorb coated with hydroxyapatite) showed clinical, radiographic, and histologic evidence of increased healing compared with control. The addition of hydroxyapatite did not augment healing.We concluded that the healing of critical-size defects in rabbits can be augmented by the addition of Lactosorb membranes. There is no evidence that platelet gel augments the healing of critical defects in rabbits.

An Experimental Design to Study Adipocyte Stem Cells for Reconstruction of Calvarial Defects

Autogenous osteogenic differentiated rat adipose stromal cells (ASCs) were used to repopulate cadaveric processed bone in a critical calvarial defect model to improve bone healing as compared with the standard treatment of cadaveric bone implantation alone. Forty-two skeletally mature Sprague-Dawley rats were randomized to 5 groups including bone graft only, bone/osteogenic differentiated ASCs, fibrin glue/osteogenic differentiated ASCs, and bone/fibrin glue/osteogenic differentiated ASCs; 2 animal calvarias were left empty. Adipose stromal cells were isolated from the inguinal fat pad of rats and differentiated into osteogenic cells, verified using von Kossa and alkaline phosphatase staining, and osteocalcin immunohistochemistry. These cells were added to sterilized, 8-mm cadaveric bone graft disks and placed back into calvarial defect for 6 weeks. The rat calvarias then underwent bone density analysis and histology. Intact cells were observed in the bone graft of the bone/osteogenic differentiated ASC group only. Islands of bone were seen in the bone-graft-only group, the bone/osteogenic differentiated ASC group, and the bone/fibrin/osteogenic differentiated ASC group. The bone-graft-only group and bone/osteogenic differentiated ASC group were similar in bone mineral density (1397 +/- 184.5 vs 1365 +/- 160.4). The bone/fibrin/osteogenic differentiated ASC group density was less than the bone and bone/osteogenic differentiated ASC groups at 835.2 +/- 319.5 (P < 0.001). Allograft bone scaffolds with autogenous osteogenic differentiated ASCs showed cellularity within the bone grafts and had larger bone islands. The presence of osteogenic differentiated ASCs did not increase overall bone density compared with bone graft only.

Bone regeneration of critical calvarial defect in goat model by PLGA/TCP/rhBMP-2 scaffolds prepared by low-temperature rapid-prototyping technology

Active artificial bone composed of poly lactide-co-glycolide (PLGA)/ tricalcium phosphate (TCP) was prefabricated using low-temperature rapid-prototyping technology so that the process of osteogenesis could be observed in it. PLGA and TCP were the primary materials, they were molded at low temperature, then recombinant human bone morphogenetic protein-2 (rhBMP-2) was added to form an active artificial bone. Goats with standard cranial defects were randomly divided into experimental (implants with rhBMP-2 added) and control (implants without rhBMP-2) groups, and osteogenesis was observed and evaluated by imaging and biomechanical and histological examinations. The PLGA-TCP artificial bone scaffold (90% porosity) had large and small pores of approximately 360 μm and 3–5 μm diameter. Preliminary and complete repair of the cranial defect in the goats occurred 12 and 24 weeks after surgery, respectively. The three-point bending strength of the repaired defects attained that of the normal cranium. In conclusion, low-temperature rapid-prototyping technology can preserve the biological activity of this scaffold material. The scaffold has a good three-dimensional structure and it becomes an active artificial bone after loading with rhBMP-2 with a modest degradation rate and excellent osteogenesis in the goat.

Bone Regenerative Property of Octacalcium Phosphate in Mouse Critical Sized Calvarial Defects

Bone regeneration by calcium phosphates has been known to be intricately dependent on material properties or implanted milieu of host animals, such as site and species. Critical sized calvarial defects of mouse were recently used as the model for investigating bone regeneration ability and the mechanisms. The purpose of the present study is to investigate whether the critical sized mouse calvarial defects can be utilized to examine bone regeneration with synthetic octacalcium phosphate (OCP). OCP , prepared by wet synthesis methods, was sieved 0.3 ~ 0.5 mm in diameter and used for the animal experiment. At 14 days after surgery, histological examination showed that implantation of OCP grafted defects significantly enhanced bone formation compared with the control defect. OCP tended to convert to hydroxyapatite with time. The tartrate-resistant acid phosphatase (TRAP) positive osteoclastic cells were observed around the OCP particles. The results suggest that the mouse critical sized calvarial bone defects are useful model to investigate the bone formation by the OCP implantation.

Oncostatin‐M Enhances Osteoinduction in a Rabbit Critical Calvarial Defect Model

Oncostatin-M (OSM) is a member of the interleukin-6 family of cytokines with controversial roles in bone homeostasis. Evidence supports a role in bone regulation, but the balance between healing promotion and acceleration of bone destruction is unclear. It is also uncertain as to whether these varied responses may be dose dependent or related to interactions with other growth factors within the bone microenvironment. To determine whether OSM enhances osteoinduction in a rabbit critical calvarial defect model and whether there is a dose response curve. OSM enhances osteoinduction, and there is a dose response curve favoring lower doses over higher doses. Controlled animal study using arms of increasing concentrations of OSM in an inactive demineralized bone matrix (DBM) carrier to assess the degree of osteoinduction through standard histomorphometric analysis and a variant of the radiodensitometry technique. Twenty-five skeletally mature New Zealand white rabbits were randomized into control and experimental arms. Incremental doses of OSM (30 microg, 100 microg, and 300 microg/g) in an inactivated guanidine-extracted DBM (Gu-DBM) carrier were implanted into a critically sized (13 mm) calvarial defect. Arms of carrier alone and no carrier served as controls. The animals were sacrificed at 4 weeks, and histomorphometry and radiodensitometry analyses were then performed. All OSM arms showed a statistically significant increase in bone formation and bone density compared with either control arm. There was also a statistically significant increase in bone area by histomorphometry between each OSM group, showing an inverse relationship to dose. Radiodensitometry analysis confirmed a significant bone density difference when comparing experimental groups with controls and also showed a significant difference between the low dose and the higher doses of OSM. It failed to show any significance between the higher two doses when compared with each other. OSM enhances osteoinduction in vivo and will close a critically sized calvarial defect in a rabbit model when delivered in a Gu-DBM carrier. There appears to be an inverse dose relationship with new bone formation.

Comparison among Four Kinds of Bone Tissue Engineering Scaffold

Bone tissue engineering is a promising way to repair of bone defects. To choose a proper scaffold is still a disputable problem in bone tissue engineering. This study aimed to compare the effects of repairing critical calvarial defects with the compounds of autogenous bone marrow stromal cells (BMSCs) and coral hydroxyapatite(CHA), hydroxyapatite/ tricalcium phosphate (HA/TCP), poly(lactide-co-glycolide) (PLGA) and alginate (AG). The results showed that CHA and AG were satisfactory bone tissues engineering scaffolds among the four kinds of materials. BMSCs/CHA and BMSCs/AG are promising techniques for reconstruction of bone defects.

Stimulation of bone healing by transforming growth factor-beta 1 released from polymeric or ceramic implants.

The ability to transforming growth factor-beta 1 (TGF-beta 1), to stimulate bone healing was evaluated in a rat critical calvarial defect model. Both a low dose and a high dose of TGF-beta 1 were incorporated into two different types of implants: one made from a composite of poly(lactic-co-glycolic acid) (PLPG) (50:50) and demineralized bone matrix (DBM), and the other from calcium sulfate (CaSO 4). Scanning electron microscopy showed that the CaSO 4 implants were more porous than the PLPG/DBM samples. Both types of implants released biologically active TGF-beta 1 for over 300 h in vitro. The samples were implanted in a 9-mm diameter rat calvarial defect for 6 weeks along with contralateral control implants containing no TGF-beta 1. Microradiography and histological analysis were used to assess the bone healing in the defects. Microradiography revealed that the greatest amount of calcified bone (67.5%) was present in in the CaSO 4 implants containing a high dose of TGF-beta 1 while minimal new bone formation occurred in the PLPG/DBM implants. Histologically, the PLPG/DBM implants exhibited an inflammatory response with little mineralization or bone formation. The defects containing the PLPG/DBM implants consisted of a connective tissue stroma with large void spaces. Giant cells and numerous polymorphonuclear leukocytes were present throughout the implants. In contrast, the CaSO 4 implants had only a few inflammatory cells and the presence of mineralization and true bone was a more consistent feature. These preliminary studies show that TGF-beta 1 is capable of inducing new bone formation. Furthermore, the materials used to deliver the growth factor can play a significant role in the bone healing process.