Critical Bone Defects Research Articles

Visualizing Angiogenesis by Multiphoton Microscopy In Vivo in Genetically Modified 3D-PLGA/nHAp Scaffold for Calvarial Critical Bone Defect Repair.

The reconstruction of critically sized bone defects remains a serious clinical problem because of poor angiogenesis within tissue-engineered scaffolds during repair, which gives rise to a lack of sufficient blood supply and causes necrosis of the new tissues. Rapid vascularization is a vital prerequisite for new tissue survival and integration with existing host tissue. The de novo generation of vasculature in scaffolds is one of the most important steps in making bone regeneration more efficient, allowing repairing tissue to grow into a scaffold. To tackle this problem, the genetic modification of a biomaterial scaffold is used to accelerate angiogenesis and osteogenesis. However, visualizing and tracking in vivo blood vessel formation in real-time and in three-dimensional (3D) scaffolds or new bone tissue is still an obstacle for bone tissue engineering. Multiphoton microscopy (MPM) is a novel bio-imaging modality that can acquire volumetric data from biological structures in a high-resolution and minimally-invasive manner. The objective of this study was to visualize angiogenesis with multiphoton microscopy in vivo in a genetically modified 3D-PLGA/nHAp scaffold for calvarial critical bone defect repair. PLGA/nHAp scaffolds were functionalized for the sustained delivery of a growth factor pdgf-b gene carrying lentiviral vectors (LV-pdgfb) in order to facilitate angiogenesis and to enhance bone regeneration. In a scaffold-implanted calvarial critical bone defect mouse model, the blood vessel areas (BVAs) in PHp scaffolds were significantly higher than in PH scaffolds. Additionally, the expression of pdgf-b and angiogenesis-related genes, vWF and VEGFR2, increased correspondingly. MicroCT analysis indicated that the new bone formation in the PHp group dramatically improved compared to the other groups. To our knowledge, this is the first time multiphoton microscopy was used in bone tissue-engineering to investigate angiogenesis in a 3D bio-degradable scaffold in vivo and in real-time.

Visualizing Angiogenesis by Multiphoton Microscopy<em> In Vivo</em> in Genetically Modified 3D-PLGA/nHAp Scaffold for Calvarial Critical Bone Defect Repair

The reconstruction of critically sized bone defects remains a serious clinical problem because of poor angiogenesis within tissue-engineered scaffolds during repair, which gives rise to a lack of sufficient blood supply and causes necrosis of the new tissues. Rapid vascularization is a vital prerequisite for new tissue survival and integration with existing host tissue. The de novo generation of vasculature in scaffolds is one of the most important steps in making bone regeneration more efficient, allowing repairing tissue to grow into a scaffold. To tackle this problem, the genetic modification of a biomaterial scaffold is used to accelerate angiogenesis and osteogenesis. However, visualizing and tracking in vivo blood vessel formation in real-time and in three-dimensional (3D) scaffolds or new bone tissue is still an obstacle for bone tissue engineering. Multiphoton microscopy (MPM) is a novel bio-imaging modality that can acquire volumetric data from biological structures in a high-resolution and minimally-invasive manner. The objective of this study was to visualize angiogenesis with multiphoton microscopy in vivo in a genetically modified 3D-PLGA/nHAp scaffold for calvarial critical bone defect repair. PLGA/nHAp scaffolds were functionalized for the sustained delivery of a growth factor pdgf-b gene carrying lentiviral vectors (LV-pdgfb) in order to facilitate angiogenesis and to enhance bone regeneration. In a scaffold-implanted calvarial critical bone defect mouse model, the blood vessel areas (BVAs) in PHp scaffolds were significantly higher than in PH scaffolds. Additionally, the expression of pdgf-b and angiogenesis-related genes, vWF and VEGFR2, increased correspondingly. MicroCT analysis indicated that the new bone formation in the PHp group dramatically improved compared to the other groups. To our knowledge, this is the first time multiphoton microscopy was used in bone tissue-engineering to investigate angiogenesis in a 3D bio-degradable scaffold in vivo and in real-time.

Collagenous matrix as a predictor for bone formation: a digital technique for collagen quantification

The collagenous matrix plays a fundamental role in the process of bone regeneration, so it is essential to study
 how it is primarily formed in situations in which critical bone defects are created. Objective: this study seeks
 to quantify the collagenous matrix formed in critical bone defects in the calvaria of mice over the process
 of bone regeneration promoted by the association of poly(lactide-co-glycolide) (PLGA) porous scaffolds and
 stem cells from deciduous teeth (SCDT). In addition, this study attempted to establish a precise protocol for
 the digital quantification of collagen through a histological method. Materials and method: Nine Wistar rats
 were used, in which critical defects of 8.0 mm of diameter were made in their calvarium. The animals were
 divided into three groups (n = 9): I – PLGA scaffolds; II – PLGA scaffolds/SCDT; III – PLGA scaffolds/SCDT
 maintained in osteogenic medium for 13 days. Within sixty postoperative days, calvaria were removed for histometric analysis following a digital protocol. A specific digital analysis method was designed for this study, in
 which a more precise quantification and differentiation between collagen fibers and non-collagenous tissue
 was possible, excluding factors that would normally alter the results. Results: it was noted that the association
 of PLGA scaffolds and SCDT maintained in osteogenic medium resulted in collagen matrix formation statistically
 higher than the other groups (p

Effect of Resveratrol on Critical-Sized Calvarial Defects of Diabetic Rats: Histometric and Gene Expression Analysis

This study investigated the influence of resveratrol (RESV) on the repair of bone critical defects in calvaria of animals with induced diabetes mellitus (DM). One hundred rats were divided into 5 groups: induced DM+RESV administration (DM+RESV; n=20); induced DM plus placebo solution administration (DM+PLAC; n=20); induced DM plus insulin therapy (DM+INS; n=20); induced DM plus administration of RES and INS (DM+RESV+INS; n=20); and nondiabetic controls (NDM; n=20). DM was induced by intraperitoneal injection of streptozotocin 50mg/kg 3days before the surgical procedures. Two critical calvarial defects were created in each animal at the start of the study (day 0). Treatments were administered from day 0 to day 30 of the experiment, when animals were euthanized. One defect was processed for histometric analysis to measure closure of the bone defect. The tissue of the other defect was analyzed for quantification of bone morphogenetic protein-2 (BMP-2), osteopontin, osteoprotegerin, receptor activator of nuclear factor-κB ligand, runt-related transcription factor-2, osterix (Osx), β-catenin, lipoprotein receptor-related protein-5, and dikkop-1 mRNA by quantitative polymerase chain reaction. Histometric results showed that the DM+RESV, DM+RESV+INS, and NDM groups exhibited greater closure of the bone defects compared with the PLAC- or INS-treated groups (P<.05). Diabetic animals treated with RESV plus INS showed higher levels of BMP-2 and Osx; Osx also was positively increased in animals treated with INS alone (P<.05). The use of RESV, regardless of the presence of INS, positively influenced bone repair in animals with induced DM. Further, the combination of INS plus RESV was necessary for the modulation of BMP-2 gene expression.

Dicalcium Phosphate Anhydrous: An AppropriateBioceramic in Regeneration of Critical-Sized Radial Bone Defects in Rats.

The present study aimed to evaluate and compare the effectiveness of composites of calcium phosphates including β-tri calcium phosphate (β-TCP), dicalcium phosphate anhydrous (DCPA, monetite), mono-calcium phosphate monohydrate (MCPM), and hydroxyapatite (HA) with the chitosan-gelatin-platelet gel (CGP) on the healing of experimentally induced critical size radial bone defects in rats after 8weeks of injury. Eighty bilateral bone defects were created in the radial bones of 40 adult male Sprague-Dawley rats. The defects were either left empty (untreated or defect group), or treated with autograft, CGP, CGP-DCP, CGP-TCP, CGP/β-TCP/DCPA (CGP-TD), CGP-TD/MCPM (CGP-TDM), and CGP-TDM/HA (CGP-TDMH) scaffolds. The injured forelimbs were evaluated by radiography, gross morphology, three-dimensional computed tomography scanning, histopathology, histomorphometry, scanning electron microscopy, and biomechanical testing. The materials were analyzed using X-ray diffraction to verify the crystalline nature of their structures, and their crystallinity was revealed based on the diffraction peaks achieved from the XRD analysis. The best results were achieved by the CGP-DCP scaffold and the autograft. The CGP-TCP and CGP-TDMH scaffolds were not degraded, while the CGP-DCP, CGP-TDM, CGP-TD, and CGP scaffolds were biodegraded and enhanced bone formation compared with the CGP-TCP and CGP-TDMH groups (P<0.05). Overall, the CGP-DCP treated defects showed significant improvement in bone formation and union, bone volume, maximum load, and stiffness compared to the CGP group (P<0.05). It could be concluded that the CGP-DCP scaffold can be considered as a suitable substitute to autograft. In fact, this study demonstrated that DCPA or monetite has high healing potential due to its biocompatibility, biodegradability and biomechanical, osteoconductive and osteoinductive properties of this bioceramic.

Enhanced bone repair induced by human adipose-derived stem cells on osteogenic extracellular matrix ornamented small intestinal submucosa.

Our aim was to design an osteogenic extracellular matrix (ECM) coated bioscaffold and to apply it to critical bone defect repair with adipose-derived stem cells (ADSCs). Morphology of scaffolds was scanned by scanning electron microscope. Cell adhesion, proliferation and osteogenic differentiation of ADSCs on ECM-small intestinal submucosa (SIS) were evaluated by immunofluorescences staining, cell counting kit-8 and real-time qPCR, respectively. A mouse calvarial defect model was used to assess effects on bone regeneration in vivo. Abundant ECM was coated on SIS, which facilitated cell adhesion and proliferation of ADSCs. ECM-SIS induced osteogenic differentiation of ADSCs even without osteogenic inductive factors. Bone regeneration in vivo was enhanced by ECM-SIS + ADSCs via BMP/SMAD pathway. This work suggested a biofabricated SIS scaffold coated with osteogenic ECM-facilitated bone regeneration with ADSCs synergistically.

Staged reconstruction of diaphyseal fractures with segmental defects: Surgical and patient-reported outcomes

IntroductionTwo-stage limb reconstruction is an option for patients with critical size segmental bone defects following acute trauma or non-union. Reconstruction is technically demanding and associated with a high complication rate. Current protocols for limb reconstruction have well-documented challenges, and no study has reported on patient outcomes using a validated questionnaire. In this study, we aimed to examine the clinical and patient-centered outcomes following our surgical protocol for two-stage limb reconstruction following critical size segmental defects. Patients and methodsA single surgeon performed reconstruction of long bone defects using antibiotic impregnated cement spacers and intramedullary cancellous bone autograft. A retrospective chart review was performed. Three reviewers independently measured time to union based on radiographs. The Lower Extremity Functional Scale (LEFS) survey was administered to patients after most recent follow-up. ResultsTen limbs representing nine patients were included. All patients sustained a lower extremity injury, and one patient had bilateral lower extremity injuries. Average clinical follow-up was 18.3 months (range 7–33) from final surgical intervention, and follow-up to questionnaire administration was 28 months (range 24–37). The mean time between stages was 3.1 months. Average time to unrestricted weight-bearing was 7.9 months from Stage 1 (range 3.4–15.9) and 4.5 months from Stage 2 (range 1.1–11.6). Average time to full union was 16.7 months from Stage 1 (range 6.4–28.6) and 13.5 months from Stage 2 (range 1.8–27). Eight patients (nine limbs) participated in the LEFS survey, the average score was 53.1 (range 30–67), equating to 66% of full functionality (range 38%–84%). Complications included 5 infections, 3 non-unions, and one amputation. There was a moderate positive correlation between infection at any time point and non-union (R=0.65, p=0.03). Discussion and conclusionsOutcomes in this small patient cohort were good despite risks of complication. There is an association between infection and non-union. Further studies addressing clinical and functional outcomes will help to guide expectations for future surgeons and patients.

Histological Evaluation of Bone Repair with Hydroxyapatite: A Systematic Review.

The aim of this study was to evaluate the morphological bone response in animal experiments by applying hydroxyapatite grafts in critical and non-critical size bone defects. Current report followed the guidelines established by the Preferred Reporting Items for Systematic Reviews and Meta-Analyses. Animal experiments were selected by assessing repair of bone defects with hydroxyapatite as bone graft and with blood clot only as control. Eight articles were identified in specialized literature and included in the meta-analysis. Statistical analysis was carried out with a random-effect model (p=0.05). Subgroup analyses were further performed to investigate bone repair in critical and non-critical bone defects. Comprehensive analysis of bone repair outcome showed a statistically significant difference between hydroxyapatite and blood clot control (p<0.05). Subgroup analyses showed statistically significant difference for critical bone defects (p<0.05). No statistically significant difference was reported in non-critical bone defects (p>0.05). Although animal studies revealed a high risk of bias and results should be interpreted with caution, the literature suggests that non-critical bone defects may heal spontaneously and without the need of a bone graft. Conversely, when critical-size defects are present, the use of hydroxyapatite bone graft improves the bone repair process.

Osteoprogenitor cells can enhance early bone formation in critical bone defects in dogs

ABSTRACT: The aim of this study was to evaluate the effect of osteoprogenitor cells derived from mesenchymal stem cells from adipose tissue (OC-AD-MSCs), and differentiated into osteoblasts, in the treatment of critical bone defects in dogs. Adipose tissue derived mesenchymal stem cells (AD-MSCs) were subjected to osteogenic differentiation for 21 days and used in the treatment of bone defects in dogs radius. Either three experimental groups were bone defects treated with OC-AD-MSCs (OC), defects filled with autogenous bone (Control- C +), or empty defects (Control- C -). Bone regeneration was assessed by radiology, densitometry, and histomorphometry. The area of new bone formation was higher in the OC group compared to the control group (C-) on postoperative day 15. Defects treated with OC-AD-MSCs showed greater neovascularization than the other two groups at 90 days. We concluded that treatment with OC-AD-MSCs increased the area of new bone formation 15 days after surgery; however, it didn’t complete the bone union in critical bone defects in the radius of dogs at 90 days.

Systematic evaluation of the osteogenic capacity of low-melting bioactive glass-reinforced 45S5 Bioglass porous scaffolds in rabbit femoral defects

Due to the low strength and high brittleness of 45S5 Bioglass®, it is still a great challenge for the three-dimensional porous 45S5 Bioglass® to treat mechanically required loaded bone defects. Therefore, 45S5 Bioglass®-derived bioactive glass-ceramic (BGC) porous scaffolds were fabricated at a low temperature sintering condition with and without the addition of 4% low-melting ZnO/B2O3 (ZB) bioactive glass as a reinforcing agent and using 350- or 500 μm paraffin microspheres as a porogen. The pore structure characterization for the scaffolds indicated that the scaffolds containing 4% ZB had very good macroporous structures of ∼313 and ∼448 μm in pore size and over 70% porosity with appreciable strength (>15 MPa), which was about four times higher than that those manufactured without ZB and with 350 μm porogen scaffolds. The open porosity was decreased with the addition of 4% ZB but the interconnected pore percentage (>50 μm) was increased with increasing the porogen size from 350 to 500 μm. In vivo investigations revealed that the stronger scaffolds containing 4% ZB and 500 μm porogen were particularly beneficial for osteogenic capacity in critical size femoral bone defects, accompanied with an accelerated bone ingrowth (6–18 weeks) and the material itself experiencing mild resorption. In contrast, both the scaffolds with smaller pore sizes exhibited a low level of new bone ingrowth (<32%) after 6–12 weeks implantation. These results suggest a promising application of such 45S5 Bioglass®-derived BGC scaffolds in a clinical setting, especially for mechanically loaded bone defects.

Substance P/dexamethasone-encapsulated PLGA scaffold fabricated using supercritical fluid process for calvarial bone regeneration.

Poly(lactic-co-glycolic acid) (PLGA) scaffolds encapsulated with substance P (SP) and dexamethasone (Dex) by the supercritical CO2 foaming method were fabricated to treat calvarial bone. We compared the release profiles of SP and Dex according to the incorporation methods using encapsulation or dipping. Ninety percent of the SP or Dex molecules in the scaffolds prepared by the encapsulating method were released by day 14 or day 6, respectively. In vivo real-time assays for human mesenchymal stem cell (hMSC) tracking were performed to confirm the MSC recruitment abilities of the scaffolds. The results showed that the optical intensity of the SP-encapsulated group was 2.59 times higher than that of the phosphate-buffered saline group and 1.3 times higher than that of the SP-dipping group. Furthermore, we compared the angiogenesis activity of the scaffolds. In the SP-encapsulated group, 72.9 ± 2.6% of the vessels showed matured features by 1 week, and it increased to 82.0 ± 4.6% after 4 weeks. We implanted the scaffolds into rat calvarial defects. After 24 weeks, SP- and Dex-encapsulated scaffolds showed 67.1% and 26.2% higher bone formation than those of the Dex-encapsulated group and SP-encapsulated group, respectively, and they formed 36.1% more bone volume compared with the SP- and Dex-dipped scaffolds. Consequently, the results of this study suggest that SP- and Dex-encapsulated scaffolds made by the supercritical CO2 foaming method could be a good treatment modality to treat critical bone defects without cell transplantation by recruiting autologous stem cells and forming new bone tissues. Copyright © 2017 John Wiley & Sons, Ltd.

The Effects of Allograft Combined with Ozone Therapy on Regeneration of Calvarial Defects in Rats

Aim: Ozone accelerates wound healing and increases the blood supply to that in previous studies reported in the literature. In this study, we claim that ozone therapy can increase the bone regeneration of the combined used with bone allograft calvarial defects in rats. Material-Method: 12 male Wistar Rats were used in this study. Critical bone defects in the rat skulls were created by means of trephan bur (Mis Implant Tech, Shlomi, Israel) , opening a 5 mm diameter defect on the right side of the naso-frontal region of the skull. The rats were separated into 4 groups in random order;1-Control defect (n=6),2-Ozone application into the control defect (control+ ozone) group (n=6),3-Allograft group (n=6) 4-Ozone Combined with Allograft application (allograft + ozone) (n=6). The defects were administered at 80% density ozone for 14 days (Ozonytron, W&H, Burmoos, Austria). Rats sacrifice at 8. week Results: The osteoblast numbers were not significantly different in the control and control + ozone groups in the 8 th week .Allograft + ozone group, the osteoblast numbers increased at a statistically significant in the 8 th week when compared with the allograft group. Bone area measurements were statistically analyzed among the groups in the 8 th week, it was observed that the allograft + ozone group showed a significant increase in the bone area amount while no statistical significance was observed in the ozone-treated contol defects, and new bone formation was observed less in the control defects. Conclusions: The results of this study , allograft + ozone combination increases the osteoblast number and new bone area at an important level when compared with the allograft group alone.

A novel method for segmenting and aligning the pre- and post-implantation scaffolds of resorbable calcium-phosphate bone substitutes.

Calcium-phosphate scaffolds are being increasingly used to repair critical bone defects. Methods for the accurate characterization of the repair process are still lacking. The present study introduced and validated a novel image-processing technique, using micro-computed tomography (mCT) datasets, to investigate material phases present in biopsies. Specifically, the new method combined mCT datasets from the scaffold before and after implantation to access the characteristic data of the ceramic for more accurate analysis of bone biopsies, and as such to better understand the interactions of the scaffold design and the bone repair process.

Reconstruction of patellar tendon following implantation of proximal tibia megaprosthesis for the treatment of post-traumatic septic bone defects

IntroductionLatest advances made in joint replacement implants allows reconstruction of entire limbs. These special prostheses or megaprostheses were originally designed for the treatment of severe oncological bone loss. Nowadays, however, the indications and applications of these devices are expanding to other orthopaedic and trauma clinical conditions. Since 2008 we have implanted 152 megaprostheses in non-oncological conditions: 87 were implanted for post-traumatic failures aseptic/septic (represented by complex non-unions and critical size bone defects); 26 total femur, 52 distal femur and 9 proximal tibia. In this group of patients bone and soft tissues conditions are completely different compared to patients with oncological back ground. The presence of infection and previous surgeries can lead to adhesion, scar interference, muscular and tendon impairment and skin problems that lead to reduced function and severe joint stiffness. The purpose of this study is to evaluate the results of treatment of reconstruction of patellar tendon during implantation of proximal tibia megaprosthesis for the treatment of septic post traumatic critical bone defects. Patients and methodsIn this retrospective study, we evaluated 9 patients treated with proximal tibia megaprosthesis who underwent patellar tendon reconstruction. All patients presented a complete patellar tendon disruption at the time of prosthesis implantation. Procedures of reconstruction included a tendon-plasty of quadriceps and/or patellar tendons, a pie crusting of quadriceps fascia, a reinforcement of the apparatus with synthetic tendon graft substitutes (LARS) and a medial gastrocnemius muscular flap to reconstruct the extensor mechanism and obtain skin coverage when needed. The average follow up was 18 months (9–36). For each of the cases, we analysed the complications occurred regarding septic recurrence, patellar fracture, quadriceps and patellar tendon rupture and number of reinterventions. The clinical outcome was assessed by the WOMAC Score. Results: In all cases there was no infection recurrence or skin related problems. None of the patients require prosthesis revision due to loosening or device failure. No patellar fracture or quadriceps tendon failure was recorded.One patient presented a rupture of the reconstructed patellar tendon due to a trauma incident 18 months after the implantation and he required revision surgery. From a clinical point of view the average WOMAC score was 62.4 at 1 month rising to 72.6 at 3 months, 78.2 at 6 months, 76.4 at 1 year and 74.8 at 18 months. ConclusionWhen proximal tibia megaprosthesis is implanted and there are soft tissue and patellar tendon deficiency, soft tissue reconstruction can be achieved by appropriate lengthening of the tendon and a gastrocnemius flap reinforced by LARS. Such an approach allows restoration of the extensor mechanism and coverage of the prosthesis in an area where skin problems are frequently very common.

Retraction note: Histopathological features of bone regeneration in a canine segmental ulnar defect model

Today, finding an ideal biomaterial to treat the large bone defects, delayed unions and non-unions remains a challenge for orthopaedic surgeions and researchers. Several studies have been carried out on the subject of bone regeneration, each having its own advantages. The present study has been designed in vivo to evaluate the effects of cellular auto-transplantation of tail vertebrae on healing of experimental critical bone defect in a dog model. Six indigenous breeds of dog with 32 ± 3.6 kg average weight from both sexes (5 males and 1 female) received bilateral critical-sized ulnar segmental defects. After determining the health condition, divided to 2 groups: The Group I were kept as control I (n = 1) while in Group II (experimental group; n = 5) bioactive bone implants were inserted. The defects were implanted with either autogeneic coccygeal bone grafts in dogs with 3-4 cm diaphyseal defects in the ulna. Defects were stabilized with internal plate fixation, and the control defects were not stabilized. Animals were euthanized at 16 weeks and analyzed by histopathology. Histological evaluation of this new bone at sixteen weeks postoperatively revealed primarily lamellar bone, with the formation of new cortices and normal-appearing marrow elements. And also reformation cortical compartment and reconstitution of marrow space were observed at the graft-host interface together with graft resorption and necrosis responses. Finally, our data were consistent with the osteoconducting function of the tail autograft. Our results suggested that the tail vertebrae autograft seemed to be a new source of autogenous cortical bone in order to supporting segmental long bone defects in dogs. Furthermore, cellular autotransplantation was found to be a successful replacement for the tail vertebrae allograft bone at 3-4 cm segmental defects in the canine mid- ulna. Clinical application using graft expanders or bone autotransplantation should be used carefully and requires further investigation. The virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/2028232688119271

Osteoblast-seeded bioglass/gelatin nanocomposite: a promising bone substitute in critical-size calvarial defect repair in rat.

Amid the plethora of methods to repair critical bone defects, there is no one perfect approach. In this study, we sought to evaluate a potent 3-dimensional (3D) bioactive SiO2-CaO-P2O5 glasses (bioglass)/gelatin (gel) scaffold for its biocompatibility by seeding cells as well as for its regenerative properties by animal implantation. Osteoblast cells were seeded onto nanocomposite scaffolds to investigate the process of critical-size calvarial defect via new bone formation. Scanning electron microscopy (SEM) was used to validate topography of the scaffolds, its homogeneity and ideal cellular attachment. Proliferation assay and confocal microscopy were used to evaluate its biocompatibility. To validate osteogenesis of the bioactive nanocomposite scaffolds, they were first implanted into rats and later removed and analyzed at different time points post mortem using histological, immunohistochemical and histomorphometric methods. Based on in vitro results, we showed that our nanocomposite is highly cell-compatible material and allows for osteoblasts to adhere, spread and proliferate. In vivo results indicate that our nanocomposite provides a significant contribution to bone regeneration and is highly biodegradable and biocompatible. So, seeded scaffolds with osteoblasts enhanced repair of critical bone defects via osteogenesis. We demonstrate the feasibility of engineering a nanocomposite scaffold with an architecture resembling the human bone, and provide proof-of-concept validation for our scaffold using a rat animal model.

Acceleration of critical bone defect healing by guided low-intensity ultrasound in a rat tibial model

Bone defects, caused by trauma, inflammation, and tumors, may lead to loss of limb functions and substantially affect the quality of patients’ life. Low-intensity ultrasound (LIUS) can transmit through and into living tissues as acoustic pressure waves, and generate dynamic acoustic radiation force in the local region to induce stem cell recruitment, and mineralization. The aim of this study was to evaluate the effect of LIUS induced healing in a rat tibial critical defect. A total of 40 skeletally mature female Lewis rats were used in this study with a surgical bone defects of 2 mm in-diameter at proximal tibia. The defects of the left tibia (LIUS group) received daily treatment of ultrasound, and the defects in the right tibia (Control) received sham stimulation. A modified repetitive frequency at 100 Hz, characteristic frequency of 1 MHz at an intensity of 30 mW/cm2 were used for the stimulation for 20-min/day. After 2 weeks, the μCT images showed that trabecular bone formed in the defect area of both LIUS and control groups. LIUS treated tibia showed more increased in BV/TV, Cont.D, Tb.N, and BMD, and significant decrease in Tb.Sp compared to control. Four-point bending testing showed LIUS treatment significantly increased the bone stiffness (kN/mm) relative to control group. The histological analysis and μCT data revealed that LIUS therapy can significantly increase new trabecular bone volume, so as the stiffness and ultimate stress of LIUS treated group with two-week as optimized treatment period for healing.

Investigation of angiogenesis by in vivo multiphoton microscopy during bone formation in murine calvarial critical bone defect repaired by genetically modified 3D-PLGA/nHAp SCAFFOLD

Investigation of angiogenesis by in vivo multiphoton microscopy during bone formation in murine calvarial critical bone defect repaired by genetically modified 3D-PLGA/nHAp SCAFFOLD

Mandibular Jaw Bone Regeneration Using Human Dental Cell-Seeded Tyrosine-Derived Polycarbonate Scaffolds.

Here we present a new model for alveolar jaw bone regeneration, which uses human dental pulp cells (hDPCs) combined with tyrosine-derived polycarbonate polymer scaffolds [E1001(1k)] containing beta-tricalcium phosphate (β-TCP) [E1001(1k)/β-TCP]. E1001(1k)/β-TCP scaffolds (5 mm diameter × 1 mm thickness) were fabricated to fit a 5 mm rat mandibular ramus critical bone defect. Five experimental groups were examined in this study: (1) E1001(1k)/β-TCP scaffolds seeded with a high density of hDPCs, 5.0 × 10(5) hDPCs/scaffold (CH); (2) E1001(1k)/β-TCP scaffolds seeded with a lower density of hDPCs, 2.5 × 10(5) hDPCs/scaffold (CL); (3) acellular E1001(1k)/β-TCP scaffolds (SA); (4) acellular E1001(1k)/β-TCP scaffolds supplemented with 4 μg recombinant human bone morphogenetic protein-2 (BMP); and (5) empty defects (EDs). Replicate hDPC-seeded and acellular E1001(1k)/β-TCP scaffolds were cultured in vitro in osteogenic media for 1 week before implantation for 3 and 6 weeks. Live microcomputed tomography (μCT) imaging at 3 and 6 weeks postimplantation revealed robust bone regeneration in the BMP implant group. CH and CL groups exhibited similar uniformly distributed mineralized tissue coverage throughout the defects, but less than the BMP implants. In contrast, SA-treated defects exhibited sparse areas of mineralized tissue regeneration. The ED group exhibited slightly reduced defect size. Histological analyses revealed no indication of an immune response. In addition, robust expression of dentin and bone differentiation marker expression was observed in hDPC-seeded scaffolds, whereas, in contrast, BMP and SA implants exhibited only bone and not dentin differentiation marker expression. hDPCs were detected in 3-week but not in 6-week hDPC-seeded scaffold groups, indicating their survival for at least 3 weeks. Together, these results show that hDPC-seeded E1001(1k)/β-TCP scaffolds support the rapid regeneration of osteo-dentin-like mineralized jaw tissue, suggesting a promising new therapy for alveolar jaw bone repair and regeneration.

Investigation of angiogenesis in bioactive 3-dimensional poly(d,l-lactide-co-glycolide)/nano-hydroxyapatite scaffolds by in vivo multiphoton microscopy in murine calvarial critical bone defect

Reconstruction of critical size bone defects remains a major clinical challenge because of poor bone regeneration, which is usually due to poor angiogenesis during repair. Satisfactory vascularization is a prerequisite for the survival of grafts and the integration of new tissue with existing tissue. In this work, we investigated angiogenesis in 3D scaffolds by in vivo multiphoton microscopy during bone formation in a murine calvarial critical bone defect model and evaluated bone regeneration 8weeks post-implantation. The continuous release of bioactive lentiviral vectors (LV-pdgfb) from the scaffolds could be detected for 5days in vitro. In vivo, the released LV-pdgfb transfected adjacent cells and expressed PDGF-BB, facilitating angiogenesis and enhancing bone regeneration. The expression of both pdgfb and the angiogenesis-related genes vWF and VEGFR2 was significantly increased in the pdgfb gene-carrying scaffold (PHp) group. In addition, microCT scanning and histomorphology results proved that there was more new bone ingrowth in the PHp group than in the PLGA/nHA (PH) and control groups. MicroCT parameters, including BMD, BV/TV, Tb.Sp, and Tb.N indicated that there was significantly more new bone formation in the PHp group than in the other groups. With regard to neovascularization, 8weeks post-implantation, blood vessel areas (BVAs) were 9428±944μm2, 4090±680.3μm2, and none in the PHp, PH, and control groups, respectively. At each time point, BVAs in the PHp scaffolds were significantly higher than in the PH scaffolds. To our knowledge, this is the first use of multiphoton microscopy in bone tissue-engineering to investigate angiogenesis in scaffolds in vivo. This method represents a valuable tool for investigating neovascularization in bone scaffolds to determine if a certain scaffold is beneficial to neovascularization. We also proved that delivery of the pdgfb gene alone can improve both angiogenesis and bone regeneration Acronyms. Statement of SignificanceReconstruction of critical size bone defects remains a major clinical challenge because of poor bone regeneration, which is usually due to poor angiogenesis during repair. Satisfactory vascularization is a prerequisite for the survival of grafts and the integration of new tissue with existing tissue. In this work, we investigated angiogenesis in 3D scaffolds by in vivo multiphoton microscopy during bone formation in a murine calvarial critical bone defect model and evaluated bone regeneration 8weeks post-implantation. To verify that pdgfb-expressing vectors carried by the scaffolds can promote angiogenesis in 3D-printed scaffolds in vivo, we monitored angiogenesis within the implants by multiphoton microscopy. To our knowledge, this is the first study to dynamically investigate angiogenesis in bone tissue engineering scaffolds in vivo.