A bioactive synthetic membrane improves bone healing in a preclinical nonunion model.
OBJECTIVES: High energy long bone fractures with critical bone loss are at risk for nonunion without strategic intervention. We hypothesize that a synthetic membrane implanted at a single stage improves bone healing in a preclinical nonunion model.METHODS: Using standard laboratory techniques, microspheres encapsulating bone morphogenic protein-2 (BMP2) or platelet derived growth factor (PDGF) were designed and coupled to a type 1 collagen sheet. Critical femoral defects were created in rats and stabilized by locked retrograde intramedullary nailing. The negative control group had an empty defect. The induced membrane group (positive control) had a polymethylmethacrylate spacer inserted into the defect for four weeks and replaced with a bare polycaprolactone/beta-tricalcium phosphate (PCL/beta-TCP) scaffold at a second stage. For the experimental groups, a bioactive synthetic membrane embedded with BMP2, PDGF or both enveloped a PCL/beta-TCP scaffold was implanted in a single stage. Serial radiographs were taken at 1, 4, 8, and 12 weeks postoperatively from the definitive procedure and evaluated by two blinded observers using a previously described scoring system to judge union as primary outcome.RESULTS: All experimental groups demonstrated better union than the negative control (p=0.01). The groups with BMP2 incorporated into the membrane demonstrated higher average union scores than the other groups (p=0.01). The induced membrane group performed similarly to the PDGF group. Complete union was only demonstrated in groups with BMP2-eluting membranes.CONCLUSIONS: A synthetic membrane comprised of type 1 collagen embedded with controlled release BMP2 improved union of critical bone defects in a preclinical nonunion model.
View details for DOI 10.1016/j.injury.2022.01.015
View details for PubMedID 35078617
Applying Deep Learning to Quantify Empty Lacunae in Histologic Sections of Osteonecrosis of the Femoral Head.
Journal of orthopaedic research : official publication of the Orthopaedic Research Society
Osteonecrosis of the femoral head (ONFH) is a disease in which inadequate blood supply to the subchondral bone causes death of cells in the bone marrow. Decalcified histology and assessment of the percentage of empty lacunae are used to quantify the severity of ONFH. However, the current clinical practice of manually counting cells is a tedious and inefficient process. We utilized the power of artificial intelligence by training an established deep convolutional neural network framework, Faster-RCNN, to automatically classify and quantify osteocytes (healthy and pyknotic) and empty lacunae in 135 histology images. The adjusted correlation coefficient between the trained cell classifier and the ground truth was R = 0.98. The methods detailed in this work significantly reduced the manual effort of cell counting in ONFH histological samples and can be translated to other fields of image quantification. This article is protected by copyright. All rights reserved.
View details for DOI 10.1002/jor.25201
View details for PubMedID 34676596
The effect of genetically modified platelet-derived growth factor-BB over-expressing mesenchymal stromal cells during core decompression for steroid-associated osteonecrosis of the femoral head in rabbits.
Stem cell research & therapy
2021; 12 (1): 503
BACKGROUND: Approximately one third of patients undergoing core decompression (CD) for early-stage osteonecrosis of the femoral head (ONFH) experience progression of the disease, and subsequently require total hip arthroplasty (THA). Thus, identifying adjunctive treatments to optimize bone regeneration during CD is an unmet clinical need. Platelet-derived growth factor (PDGF)-BB plays a central role in cell growth and differentiation. The aim of this study was to characterize mesenchymal stromal cells (MSCs) that were genetically modified to overexpress PDGF-BB (PDGF-BB-MSCs) in vitro and evaluate their therapeutic effect when injected into the bone tunnel at the time of CD in an in vivo rabbit model of steroid-associated ONFH.METHODS: In vitro studies: Rabbit MSCs were transduced with a lentivirus vector carrying the human PDGF-BB gene under the control of either the cytomegalovirus (CMV) or phosphoglycerate (PGK) promoter. The proliferative rate, PDGF-BB expression level, and osteogenic differentiation capacity of unmodified MSCs, CMV-PDGF-BB-MSCs, and PGK-PDGF-BB-MSCs were assessed. In vivo studies: Twenty-four male New Zealand white rabbits received an intramuscular (IM) injection of methylprednisolone 20mg/kg. Four weeks later, the rabbits were divided into four groups: the CD group, the hydrogel [HG, (a collagen-alginate mixture)] group, the MSC group, and the PGK-PDGF-BB-MSC group. Eight weeks later, the rabbits were sacrificed, their femurs were harvested, and microCT, mechanical testing, and histological analyses were performed.RESULTS: In vitro studies: PGK-PDGF-BB-MSCs proliferated more rapidly than unmodified MSCs (P<0.001) and CMV-PDGF-BB-MSCs (P<0.05) at days 3 and 7. CMV-PDGF-BB-MSCs demonstrated greater PDGF-BB expression than PGK-PDGF-BB-MSCs (P<0.01). However, PGK-PDGF-BB-MSCs exhibited greater alkaline phosphatase staining at 14days (P<0.01), and osteogenic differentiation at 28days (P=0.07) than CMV-PDGF-BB-MSCs. In vivo: The PGK-PDGF-BB-MSC group had a trend towards greater bone mineral density (BMD) than the CD group (P=0.074). The PGK-PDGF-BB-MSC group demonstrated significantly lower numbers of empty lacunae (P<0.001), greater osteoclast density (P<0.01), and greater angiogenesis (P<0.01) than the other treatment groups.CONCLUSION: The use of PGK-PDGF-BB-MSCs as an adjunctive treatment with CD may reduce progression of osteonecrosis and enhance bone regeneration and angiogenesis in the treatment of early-stage ONFH.
View details for DOI 10.1186/s13287-021-02572-7
View details for PubMedID 34526115
The efficacy of lapine preconditioned or genetically modified IL4 over-expressing bone marrow-derived mesenchymal stromal cells in corticosteroid-associated osteonecrosis of the femoral head in rabbits.
2021; 275: 120972
Cell-based therapy for augmentation of core decompression (CD) using mesenchymal stromal cells (MSCs) is a promising treatment for early stage osteonecrosis of the femoral head (ONFH). Recently, the therapeutic potential for immunomodulation of osteogenesis using preconditioned (with pro-inflammatory cytokines) MSCs (pMSCs), or by the timely resolution of inflammation using MSCs that over-express anti-inflammatory cytokines has been described. Here, pMSCs exposed to tumor necrosis factor-alpha and lipopolysaccharide for 3 days accelerated osteogenic differentiation in vitro. Furthermore, injection of pMSCs encapsulated with injectable hydrogels into the bone tunnel facilitated angiogenesis and osteogenesis in the femoral head in vivo, using rabbit bone marrow-derived MSCs and a model of corticosteroid-associated ONFH in rabbits. In contrast, in vitro and in vivo studies demonstrated that genetically-modified MSCs that over-express IL4 (IL4-MSCs), established by using a lentiviral vector carrying the rabbit IL4 gene under the cytomegalovirus promoter, accelerated proliferation of MSCs and decreased the percentage of empty lacunae in the femoral head. Therefore, adjunctive cell-based therapy of CD using pMSCs and IL4-MSCs may hold promise to heal osteonecrotic lesions in the early stage ONFH. These interventions must be applied in a temporally sensitive fashion, without interfering with the mandatory acute inflammatory phase of bone healing.
View details for DOI 10.1016/j.biomaterials.2021.120972
View details for PubMedID 34186237
Effect of porosity of a functionally-graded scaffold for the treatment of corticosteroid-associated osteonecrosis of the femoral head in rabbits.
Journal of orthopaedic translation
2021; 28: 90–99
Background/Objective: Core decompression (CD) with scaffold and cell-based therapies is a promising strategy for providing both mechanical support and regeneration of the osteonecrotic area for early stage osteonecrosis of the femoral head (ONFH). We designed a new 3D printed porous functionally-graded scaffold (FGS) with a central channel to facilitate delivery of transplanted cells in a hydrogel to the osteonecrotic area. However, the optimal porous structural design for the FGS for the engineering of bone in ONFH has not been elucidated. The aim of this study was to fabricate and evaluate two different porous structures (30% or 60% porosity) of the FGSs in corticosteroid-associated ONFH in rabbits.Methods: Two different FGSs with 30% or 60% porosity containing a 1-mm central channel were 3D printed using polycaprolactone and beta-tricalcium phosphate. The FGS was 3-mm diameter and 32-mm length and was composed of three segments: 1-mm in length for the non-porous proximal segment, 22-mm in length for the porous (30% versus 60%) middle segment, and 9-mm in length for the 15% porous distal segment. Eighteen male New Zealand White rabbits were given a single dose of 20mg/kg methylprednisolone acetate intramuscularly. Four weeks later, rabbits were divided into three groups: the CD group, the 30% porosity FGS group, and the 60% porosity FGS group. In the CD group, a 3-mm diameter drill hole was created into the left femoral head. In the FGS groups, a 30% or 60% porosity implant was inserted into the bone tunnel. Eight weeks postoperatively, femurs were harvested and microCT, mechanical, and histological analyses were performed.Results: The actual porosity and pore size of the middle segments were 26.4%±2.3% and 699±56mum in the 30% porosity FGS, and 56.0%±4.5% and 999±71mum in the 60% porosity FGS, respectively using microCT analysis. Bone ingrowth ratio in the 30% porosity FGS group was 73.9%±15.8%, which was significantly higher than 39.5%±13.0% in the CD group on microCT (p<0.05). Bone ingrowth ratio in the 60% porosity FGS group (61.3%±30.1%) showed no significant differences compared to the other two groups. The stiffness at the bone tunnel site in the 30% porosity FGS group was 582.4±192.3N/mm3, which was significantly higher than 338.7±164.6N/mm3 in the 60% porosity FGS group during push-out testing (p<0.05). Hematoxylin and eosin staining exhibited thick and mature trabecular bone around the porous FGS in the 30% porosity FGS group, whereas thinner, more immature trabecular bone was seen around the porous FGS in the 60% porosity FGS group.Conclusion: These findings indicate that the 30% porosity FGS may enhance bone regeneration and have superior biomechanical properties in the bone tunnel after CD in ONFH, compared to the 60% porosity FGS.Translation potential statement: The translational potential of this article: This FGS implant holds promise for improving outcomes of CD for early stage ONFH.
View details for DOI 10.1016/j.jot.2021.01.002
View details for PubMedID 33816112
Investigation of a Prevascularized Bone Graft for Large Defects in the Ovine Tibia.
Tissue engineering. Part A
In vivo bioreactors are a promising approach for engineering vascularized autologous bone grafts to repair large bone defects. In this pilot parametric study, we first developed a 3D printed scaffold uniquely designed to accommodate inclusion of a vascular bundle and facilitate growth factor delivery for accelerated vascular invasion and ectopic bone formation. Second, we established a new sheep deep circumflex iliac artery (DCIA) model as an in vivo bioreactor for engineering a vascularized bone graft and evaluated the effect of implantation duration on ectopic bone formation. Third, after 8 weeks of implantation around the DCIA, we transplanted the prevascularized bone graft to a 5 cm segmental bone defect in the sheep tibia, using the custom 3D printed BMP-2 loaded scaffold without prior in vivo bioreactor maturation as a control. Analysis by micro-computed tomography and histomorphometry found ectopic bone formation in BMP-2 loaded scaffolds implanted for 8 and 12 weeks in the iliac pouch, with greater bone formation occurring after 12 weeks. Grafts transplanted to the tibial defect supported bone growth, mainly on the periphery of the graft, but greater bone growth and less soft tissue invasion was observed in the avascular BMP-2 loaded scaffold implanted directly into the tibia without prior in vivo maturation. Histopathological evaluation noted considerably greater vascularity in the bone grafts that underwent in vivo maturation with an inserted vascular bundle compared to the avascular BMP-2 loaded graft. Our findings indicate that use of an initial DCIA in vivo bioreactor maturation step is a promising approach to developing vascularized autologous bone grafts, although scaffolds with greater osteoinductivity should be further studied.
View details for DOI 10.1089/ten.TEA.2020.0347
View details for PubMedID 33858216
Osteoinductive 3D printed scaffold healed 5cm segmental bone defects in the ovine metatarsus.
2021; 11 (1): 6704
Autologous bone grafts are considered the gold standard grafting material for the treatment of nonunion, but in very large bone defects, traditional autograft alone is insufficient to induce repair. Recombinant human bone morphogenetic protein 2 (rhBMP-2) can stimulate bone regeneration and enhance the healing efficacy of bone grafts. The delivery of rhBMP-2 may even enable engineered synthetic scaffolds to be used in place of autologous bone grafts for the treatment of critical size defects, eliminating risks associated with autologous tissue harvest. We here demonstrate that an osteoinductive scaffold, fabricated by combining a 3D printed rigid polymer/ceramic composite scaffold with an rhBMP-2-eluting collagen sponge can treat extremely large-scale segmental defects in a pilot feasibility study using a new sheep metatarsus fracture model stabilized with an intramedullary nail. Bone regeneration after 24weeks was evaluated by micro-computed tomography, mechanical testing, and histological characterization. Load-bearing cortical bridging was achieved in all animals, with increased bone volume observed in sheep that received osteoinductive scaffolds compared to sheep that received an rhBMP-2-eluting collagen sponge alone.
View details for DOI 10.1038/s41598-021-86210-5
View details for PubMedID 33758338
Development of PLGA-PEG-COOH and gelatin-based microparticles dual delivery system and E-beam sterilization effects for controlled release of BMP-2 and IGF-1.
Particle & particle systems characterization : measurement and description of particle properties and behavior in powders and other disperse systems
2020; 37 (10)
The purpose of this study was to develop a PLGA-PEG-COOH- and gelatin-based microparticles (MPs) dual delivery system for release of BMP-2 and IGF-1. We made and characterized the delivery system based on its morphology, loading capacity, Encapsulation efficiency and release kinetics. Second, we examined the effects of electron beam (EB) sterilization on BMP-2 and IGF-1 loaded MPs and their biological effects. Third, we evaluated the synergistic effect of a controlled dual release of BMP-2 and IGF-1 on osteogenesis of MSCs. Encapsulation efficiency of growth factors into gelatin and PLGA-PEG-COOH MPs are in the range of 64.78% to 76.11%. E-beam sterilized growth factor delivery systems were effective in significantly promoting osteogenesis of MSCs, although E-beam sterilization decreased the bioactivity of growth factors in MPs by approximately 22%. BMP-2 release behavior from gelatin MPs/PEG hydrogel shows a faster release (52.7%) than that of IGF-1 from the PLGA-PEG-COOH MPs/PEG hydrogel (27.3%). The results demonstrate that the gelatin and PLGA-PEG-COOH MPs based delivery system could realize temporal release of therapeutic biomolecules by incorporating different growth factors into distinct microparticles. EB sterilization was an accessible method for sterilizing growth factors loaded carriers, which could pave the way for implementing growth factor delivery in clinical applications.
View details for DOI 10.1002/ppsc.202000180
View details for PubMedID 33384477
View details for PubMedCentralID PMC7771709
- Development of PLGA-PEG-COOH and Gelatin-Based Microparticles Dual Delivery System and E-Beam Sterilization Effects for Controlled Release of BMP-2 and IGF-1 PARTICLE & PARTICLE SYSTEMS CHARACTERIZATION 2020
The Influence of Electron Beam Sterilization on In Vivo Degradation of beta-TCP/PCL of Different Composite Ratios for Bone Tissue Engineering.
2020; 11 (3)
We evaluated the effect of electron beam (E-beam) sterilization (25 kGy, ISO 11137) on the degradation of beta-tricalcium phosphate/polycaprolactone (beta-TCP/PCL) composite filaments of various ratios (0:100, 20:80, 40:60, and 60:40 TCP:PCL by mass) in a rat subcutaneous model for 24 weeks. Volumes of the samples before implantation and after explantation were measured using micro-computed tomography (micro-CT). The filament volume changes before sacrifice were also measured using a live micro-CT. In our micro-CT analyses, there was no significant difference in volume change between the E-beam treated groups and non-E-beam treated groups of the same beta-TCP to PCL ratios, except for the 0% beta-TCP group. However, the average volume reduction differences between the E-beam and non-E-beam groups in the same-ratio samples were 0.76% (0% TCP), 3.30% (20% TCP), 4.65% (40% TCP), and 3.67% (60% TCP). The E-beam samples generally had more volume reduction in all experimental groups. Therefore, E-beam treatment may accelerate degradation. In our live micro-CT analyses, most volume reduction arose in the first four weeks after implantation and slowed between 4 and 20 weeks in all groups. E-beam groups showed greater volume reduction at every time point, which is consistent with the results by micro-CT analysis. Histology results suggest the biocompatibility of TCP/PCL composite filaments.
View details for DOI 10.3390/mi11030273
View details for PubMedID 32155781