Longitudinal in vivo evaluation of bone regeneration by combined measurement of multi-pinhole SPECT and micro-CT for tissue engineering

Abstract Over the last decades, great strides were made in the development of novel implants for the treatment of bone defects. The increasing versatility and complexity of these implant designs request for concurrent advances in means to assess in vivo the course of induced bone formation in preclinical models. Since its discovery, micro-computed tomography (micro-CT) has excelled as powerful high-resolution technique for non-invasive assessment of newly formed bone tissue. However, micro-CT fails to provide spatiotemporal information on biological processes ongoing during bone regeneration. Conversely, due to the versatile applicability and cost-effectiveness, single photon emission computed tomography (SPECT) would be an ideal technique for assessing such biological processes with high sensitivity and for nuclear imaging comparably high resolution (<1 mm). Herein, we employ modular designed poly(ethylene glycol)-based hydrogels that release bone morphogenetic protein to guide the healing of critical sized calvarial bone defects. By combined in vivo longitudinal multi-pinhole SPECT and micro-CT evaluations we determine the spatiotemporal course of bone formation and remodeling within this synthetic hydrogel implant. End point evaluations by high resolution micro-CT and histological evaluation confirm the value of this approach to follow and optimize bone-inducing biomaterials.

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Erratum: Longitudinal in vivo evaluation of bone regeneration by combined measurement of multi-pinhole SPECT and micro-CT for tissue engineering

Scientific Reports ◽

10.1038/srep12391 ◽

2015 ◽

Vol 5 (1) ◽

Author(s):

Philipp S. Lienemann ◽

Stéphanie Metzger ◽

Anna-Sofia Kiveliö ◽

Alain Blanc ◽

Panagiota Papageorgiou ◽

...

Keyword(s):

Tissue Engineering ◽

Bone Regeneration ◽

Micro Ct ◽

In Vivo Evaluation ◽

Pinhole Spect

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The Effect of Autologous Platelet-Rich Plasma on Bone Regeneration by Autologous Mesenchymal Stem Cells Loaded onto Allogeneic Cancellous Bone Granules

Cells Tissues Organs ◽

10.1159/000454915 ◽

2017 ◽

Vol 203 (6) ◽

pp. 327-338 ◽

Cited By ~ 10

Author(s):

Min Wook Joo ◽

Seung Jae Chung ◽

Seung Han Shin ◽

Yang-Guk Chung

Keyword(s):

Stem Cells ◽

Bone Regeneration ◽

Cancellous Bone ◽

Platelet Rich Plasma ◽

Bone Defects ◽

Histological Evaluation ◽

Control Group ◽

Micro Ct ◽

Autologous Platelet Rich Plasma ◽

Autologous Platelet

To develop a clinically effective bone regeneration strategy, we compared bone regeneration using allogeneic cancellous bone granule scaffolds loaded with autologous bone marrow-derived mesenchymal stem cells (BM-MSC) with or without autologous platelet-rich plasma (PRP). Critical-sized segmental bone defects were made at the mid-shaft of both radiuses in 41 New Zealand White rabbits. Small-sized allogeneic cancellous bone granules (300-700 μm in diameter) loaded with BM-MSC were implanted on one side, and PRP was added. On the other side, autologous BM-MSC loaded onto allogeneic cancellous granules were grafted as a control. Bone regeneration was assessed by radiographic evaluation at 4, 8, and 16 weeks postimplantation and by micro-computed tomography (micro-CT) and histological evaluation of the retrieved specimens at 8 and 16 weeks. The experimental group did not show significantly higher bone quantity indices than the control group at any time point. Micro-CT analysis revealed that both groups had similar mean total volumes, surface areas, and other parameters at 8 and 16 weeks. Histological evaluation of 8- and 16-week specimens also showed a similar progression of new bone formation and maturation. In this experiment using a contralateral control group in the same individual, an initial single addition of PRP in allogeneic cancellous bone granules loaded with BM-MSC for critical-sized bone defects in the weight-bearing area did not induce a consequent difference in bone healing. Further research into the optimal preparation and application of PRP is necessary. Furthermore, studies involving a greater number of subjects and larger experimental animals could determine the clinical relevance of PRP treatment.

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In Vivo Bone Regeneration Induced by a Scaffold of Chitosan/Dicarboxylic Acid Seeded with Human Periodontal Ligament Cells

International Journal of Molecular Sciences ◽

10.3390/ijms20194883 ◽

2019 ◽

Vol 20 (19) ◽

pp. 4883 ◽

Cited By ~ 4

Author(s):

Teerawat Sukpaita ◽

Suwabun Chirachanchai ◽

Pornchanok Suwattanachai ◽

Vincent Everts ◽

Atiphan Pimkhaokham ◽

...

Keyword(s):

Gene Expression ◽

Bone Formation ◽

Dicarboxylic Acid ◽

Bone Regeneration ◽

Periodontal Ligament ◽

Periodontal Ligament Cells ◽

Micro Ct ◽

New Bone Formation ◽

Calvarial Defects

Chitosan/dicarboxylic acid (CS/DA) scaffold has been developed as a bone tissue engineering material. This study evaluated a CS/DA scaffold with and without seeded primary human periodontal ligament cells (hPDLCs) in its capacity to regenerate bone in calvarial defects of mice. The osteogenic differentiation of hPDLCs was analyzed by bone nodule formation and gene expression. In vivo bone regeneration was analyzed in mice calvarial defects. Eighteen mice were divided into 3 groups: one group with empty defects, one group with defects with CS/DA scaffold, and a group with defects with CS/DA scaffold and with hPDLCs. After 6 and 12 weeks, new bone formation was assessed using microcomputed tomography (Micro-CT) and histology. CS/DA scaffold significantly promoted in vitro osteoblast-related gene expression (RUNX2, OSX, COL1, ALP, and OPN) by hPDLCs. Micro-CT revealed that CS/DA scaffolds significantly promoted in vivo bone regeneration both after 6 and 12 weeks (p < 0.05). Histological examination confirmed these findings. New bone formation was observed in defects with CS/DA scaffold; being similar with and without hPDLCs. CS/DA scaffolds can be used as a bone regenerative material with good osteoinductive/osteoconductive properties.

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Integration of Epigallocatechin Gallate in Gelatin Sponges Attenuates Matrix Metalloproteinase-Dependent Degradation and Increases Bone Formation

International Journal of Molecular Sciences ◽

10.3390/ijms20236042 ◽

2019 ◽

Vol 20 (23) ◽

pp. 6042 ◽

Cited By ~ 4

Author(s):

Anqi Huang ◽

Yoshitomo Honda ◽

Peiqi Li ◽

Tomonari Tanaka ◽

Shunsuke Baba

Keyword(s):

Computed Tomography ◽

Extracellular Matrix ◽

Regenerative Medicine ◽

Bone Formation ◽

Matrix Metalloproteinase ◽

Epigallocatechin Gallate ◽

Bone Defects ◽

Micro Computed Tomography ◽

Immunohistological Staining

Matrix metalloproteinase (MMP)-2 and MMP-9 are well-known gelatinases that disrupt the extracellular matrix, including gelatin. However, the advantages of modulating MMP expression in gelatin-based materials for applications in bone regenerative medicine have not been fully clarified. In this study, we examined the effects of epigallocatechin gallate (EGCG), a major polyphenol catechin isolated from green tea, on MMP expression in gelatin sponges and its association with bone formation. Four gelatin sponges with or without EGCG were prepared and implanted into bone defects for up to 4 weeks. Histological and immunohistological staining were performed. Micro-computed tomography was used to estimate the bone-forming capacity of each sponge. Our results showed that EGCG integration attenuated MMP-2 (70.6%) and -9 expression (69.1%) in the 1 week group, increased residual gelatin (118.7%), and augmented bone formation (101.8%) in the 4 weeks group in critical-sized bone defects of rat calvaria compared with vacuum-heated gelatin sponges without EGCG. Moreover, vacuum-heated gelatin sponges with EGCG showed superior bone formation compared with other sponges. The results indicated that integration of EGCG in gelatin-based materials modulated the production and activity of MMP-2 and -9 in vivo, thereby enhancing bone-forming capacity.

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Spatio-temporal characterization of fracture healing patterns and assessment of biomaterials by time-lapsed in vivo micro-computed tomography

Scientific Reports ◽

10.1038/s41598-021-87788-6 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Esther Wehrle ◽

Duncan C. Tourolle né Betts ◽

Gisela A. Kuhn ◽

Erica Floreani ◽

Malavika H. Nambiar ◽

...

Keyword(s):

Computed Tomography ◽

Bone Regeneration ◽

Healing Process ◽

Micro Ct ◽

Micro Computed Tomography ◽

Collagen Scaffolds ◽

Non Union ◽

Spatio Temporal

AbstractThorough preclinical evaluation of functionalized biomaterials for treatment of large bone defects is essential prior to clinical application. Using in vivo micro-computed tomography (micro-CT) and mouse femoral defect models with different defect sizes, we were able to detect spatio-temporal healing patterns indicative of physiological and impaired healing in three defect sub-volumes and the adjacent cortex. The time-lapsed in vivo micro-CT-based approach was then applied to evaluate the bone regeneration potential of functionalized biomaterials using collagen and bone morphogenetic protein (BMP-2). Both collagen and BMP-2 treatment led to distinct changes in bone turnover in the different healing phases. Despite increased periosteal bone formation, 87.5% of the defects treated with collagen scaffolds resulted in non-unions. Additional BMP-2 application significantly accelerated the healing process and increased the union rate to 100%. This study further shows potential of time-lapsed in vivo micro-CT for capturing spatio-temporal deviations preceding non-union formation and how this can be prevented by application of functionalized biomaterials. This study therefore supports the application of longitudinal in vivo micro-CT for discrimination of normal and disturbed healing patterns and for the spatio-temporal characterization of the bone regeneration capacity of functionalized biomaterials.

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The combinatory effect of sinusoidal electromagnetic field and VEGF promotes osteogenesis and angiogenesis of mesenchymal stem cell-laden PCL/HA implants in a rat subcritical cranial defect

Stem Cell Research & Therapy ◽

10.1186/s13287-019-1464-x ◽

2019 ◽

Vol 10 (1) ◽

Cited By ~ 1

Author(s):

Jingyuan Chen ◽

Chang Tu ◽

Xiangyu Tang ◽

Hao Li ◽

Jiyuan Yan ◽

...

Keyword(s):

Tissue Engineering ◽

Electromagnetic Field ◽

Bone Formation ◽

Bone Regeneration ◽

Bone Defect ◽

Low Density Lipoprotein ◽

Density Lipoprotein ◽

Bone Defects ◽

Histological Evaluation ◽

Cranial Defect

Abstract Background Restoration of massive bone defects remains a huge challenge for orthopedic surgeons. Insufficient vascularization and slow bone regeneration limited the application of tissue engineering in bone defect. The effect of electromagnetic field (EMF) on bone defect has been reported for many years. However, sinusoidal EMF (SEMF) combined with tissue engineering in bone regeneration remains poorly investigated. Methods In the present study, we investigated the effect of SEMF and vascular endothelial growth factor (VEGF) on osteogenic and vasculogenic differentiation of rat bone marrow-derived mesenchymal stem cells (rBMSCs). Furthermore, pretreated rBMSC- laden polycaprolactone-hydroxyapatite (PCL/HA) scaffold was constructed and implanted into the subcritical cranial defect of rats. The bone formation and vascularization were evaluated 4 and 12 weeks after implantation. Results It was shown that SEMF and VEGF could enhance the protein and mRNA expression levels of osteoblast- and endothelial cell-related markers, respectively. The combinatory effect of SEMF and VEGF slightly promoted the angiogenic differentiation of rBMSCs. The proteins of Wnt1, low-density lipoprotein receptor-related protein 6 (LRP-6), and β-catenin increased in all inducted groups, especially in SEMF + VEGF group. The results indicated that Wnt/β-catenin pathway might participate in the osteogenic and angiogenic differentiation of rBMSCs. Histological evaluation and reconstructed 3D graphs revealed that tissue-engineered constructs significantly promoted the new bone formation and angiogenesis compared to other groups. Conclusion The combinatory effect of SEMF and VEGF raised an efficient approach to enhance the osteogenesis and vascularization of tissue-engineered constructs, which provided a useful guide for regeneration of bone defects.

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Bioglass-Incorporated Methacrylated Gelatin Cryogel for Regeneration of Bone Defects

Polymers ◽

10.3390/polym10080914 ◽

2018 ◽

Vol 10 (8) ◽

pp. 914 ◽

Cited By ~ 18

Author(s):

Song Kwon ◽

Seunghun Lee ◽

A. Sivashanmugam ◽

Janet Kwon ◽

Seung Kim ◽

...

Keyword(s):

Bone Regeneration ◽

Bone Defects ◽

Micro Ct ◽

Defect Model ◽

Cranial Defect ◽

In Vivo Testing ◽

Regeneration Of Bone ◽

Adhesion Site

Cryogels have recently gained interest in the field of tissue engineering as they inherently possess an interconnected macroporous structure. Considered to be suitable for scaffold cryogel fabrication, methacrylated gelatin (GelMA) is a modified form of gelatin valued for its ability to retain cell adhesion site. Bioglass nanoparticles have also attracted attention in the field due to their osteoinductive and osteoconductive behavior. Here, we prepare methacrylated gelatin cryogel with varying concentration of bioglass nanoparticles to study its potential for bone regeneration. We demonstrate that an increase in bioglass concentration in cryogel leads to improved mechanical property and augmented osteogenic differentiation of mesenchymal cells during in vitro testing. Furthermore, in vivo testing in mice cranial defect model shows that highest concentration of bioglass nanoparticles (2.5 w/w %) incorporated in GelMA cryogel induces the most bone formation compared to the other tested groups, as studied by micro-CT and histology. The in vitro and in vivo results highlight the potential of bioglass nanoparticles incorporated in GelMA cryogel for bone regeneration.

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Micro-Computed Tomography Analysis of Subchondral Bone Regeneration Using Osteochondral Scaffolds in an Ovine Condyle Model

Applied Sciences ◽

10.3390/app11030891 ◽

2021 ◽

Vol 11 (3) ◽

pp. 891

Author(s):

Taylor Flaherty ◽

Maryam Tamaddon ◽

Chaozong Liu

Keyword(s):

Computed Tomography ◽

Bone Regeneration ◽

Subchondral Bone ◽

Volume Ratio ◽

Bone Volume ◽

Osteochondral Defects ◽

Micro Ct ◽

Micro Computed Tomography ◽

Trabecular Thickness ◽

Osteochondral Scaffold

Osteochondral scaffold technology has emerged as a promising therapy for repairing osteochondral defects. Recent research suggests that seeding osteochondral scaffolds with bone marrow concentrate (BMC) may enhance tissue regeneration. To examine this hypothesis, this study examined subchondral bone regeneration in scaffolds with and without BMC. Ovine stifle condyle models were used for the in vivo study. Two scaffold systems (8 mm diameter and 10 mm thick) with and without BMC were implanted into the femoral condyle, and the tissues were retrieved after six months. The retrieved femoral condyles (with scaffold in) were examined using micro-computed tomography scans (micro-CT), and the micro-CT data were further analysed by ImageJ with respect to trabecular thickness, bone volume to total volume ratio (BV/TV) ratio, and degree of anisotropy of bone. Statistical analysis compared bone regeneration between scaffold groups and sub-set regions. These results were mostly insignificant (p < 0.05), with the exception of bone volume to total volume ratio when comparing scaffold composition and sub-set region. Additional trends in the data were observed. These results suggest that the scaffold composition and addition of BMC did not significantly affect bone regeneration in osteochondral defects after six months. However, this research provides data which may guide the development of future treatments.

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The Bone Regeneration Capacity of BMP-2 + MMP-10 Loaded Scaffolds Depends on the Tissue Status

Pharmaceutics ◽

10.3390/pharmaceutics13070979 ◽

2021 ◽

Vol 13 (7) ◽

pp. 979

Author(s):

Patricia Garcia-Garcia ◽

Ricardo Reyes ◽

José Antonio Rodriguez ◽

Tomas Martín ◽

Carmen Evora ◽

...

Keyword(s):

Bone Formation ◽

Bone Regeneration ◽

Growth Promotion ◽

Tissue Growth ◽

Bone Morphogenetic Protein 2 ◽

Morphogenetic Protein ◽

Therapeutic Molecules ◽

Positive Effect

Biomaterials-mediated bone formation in osteoporosis (OP) is challenging as it requires tissue growth promotion and adequate mineralization. Based on our previous findings, the development of scaffolds combining bone morphogenetic protein 2 (BMP-2) and matrix metalloproteinase 10 (MMP-10) shows promise for OP management. To test our hypothesis, scaffolds containing BMP-2 + MMP-10 at variable ratios or BMP-2 + Alendronate (ALD) were prepared. Systems were characterized and tested in vitro on healthy and OP mesenchymal stem cells and in vivo bone formation was studied on healthy and OP animals. Therapeutic molecules were efficiently encapsulated into PLGA microspheres and embedded into chitosan foams. The use of PLGA (poly(lactic-co-glycolic acid)) microspheres as therapeutic molecule reservoirs allowed them to achieve an in vitro and in vivo controlled release. A beneficial effect on the alkaline phosphatase activity of non-OP cells was observed for both combinations when compared with BMP-2 alone. This effect was not detected on OP cells where all treatments promoted a similar increase in ALP activity compared with control. The in vivo results indicated a positive effect of the BMP-2 + MMP-10 combination at both of the doses tested on tissue repair for OP mice while it had the opposite effect on non-OP animals. This fact can be explained by the scaffold’s slow-release rate and degradation that could be beneficial for delayed bone regeneration conditions but had the reverse effect on healthy animals. Therefore, the development of adequate scaffolds for bone regeneration requires consideration of the tissue catabolic/anabolic balance to obtain biomaterials with degradation/release behaviors suited for the existing tissue status.

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