Early pH Changes in Musculoskeletal Tissues upon Injury-Aerobic Catabolic Pathway Activity Linked to Inter-Individual Differences in Local pH

Local pH is stated to acidify after bone fracture. However, the time course and degree of acidification remain unknown. Whether the acidification pattern within a fracture hematoma is applicable to adjacent muscle hematoma or is exclusive to this regenerative tissue has not been studied to date. Thus, in this study, we aimed to unravel the extent and pattern of acidification in vivo during the early phase post musculoskeletal injury. Local pH changes after fracture and muscle trauma were measured simultaneously in two pre-clinical animal models (sheep/rats) immediately after and up to 48 h post injury. The rat fracture hematoma was further analyzed histologically and metabolomically. In vivo pH measurements in bone and muscle hematoma revealed a local acidification in both animal models, yielding mean pH values in rats of 6.69 and 6.89, with pronounced intra- and inter-individual differences. The metabolomic analysis of the hematomas indicated a link between reduction in tricarboxylic acid cycle activity and pH, thus, metabolic activity within the injured tissues could be causative for the different pH values. The significant acidification within the early musculoskeletal hematoma could enable the employment of the pH for novel, sought-after treatments that allow for spatially and temporally controlled drug release

Weightbearing ovine osteochondral defects heal with inadequate subchondral bone plate restoration: implications regarding osteochondral autograft harvesting

PURPOSE: It is unknown what causes donor site morbidity following the osteochondral autograft transfer procedure or how donor sites heal. Contact pressure and edge loading at donor sites may play a role in the healing process. It was hypothesized that an artificially created osteochondral defect in a weightbearing area of an ovine femoral condyle will cause osseous bridging of the defect from the upper edges, resulting in incomplete and irregular repair of the subchondral bone plate. METHODS: To simulate edge loading, large osteochondral defects were created in the most unfavourable weightbearing area of 24 ovine femoral condyles. After killing at 3 and 6 months, osteochondral defects were histologically and histomorphometrically evaluated with specific attention to subchondral bone healing and subchondral bone plate restoration. RESULTS: Osteochondral defect healing showed progressive osseous defect bridging by sclerotic circumferential bone apposition. Unfilled area decreased significantly from 3 to 6 months (P = 0.004), whereas bone content increased (n.s.). Complete but irregular subchondral bone plate restoration occurred in ten animals. In fourteen animals, an incomplete subchondral bone plate was found. Further common findings included cavitary lesion formation, degenerative cartilage changes and cartilage and subchondral bone collapse. CONCLUSIONS: Osteochondral defect healing starts with subchondral bone plate restoration. However, after 6 months, incomplete or irregular subchondral bone plate restoration and subsequent failure of osteochondral defect closure is common. Graft harvesting in the osteochondral autograft transfer procedure must be viewed critically, as similar changes are also present in humans. LEVEL OF EVIDENCE: Prognostic study, Level III

Establishment of a preclinical ovine screening model for the investigation of bone tissue engineering strategies in cancellous and cortical bone defects

Background New tissue engineering strategies for bone regeneration need to be investigated in a relevant preclinical large animal model before making the translation into human patients. Therefore, our interdisciplinary group established a simplified large animal screening model for intramembranous bone defect regeneration in cancellous and cortical bone. Methods Related to a well-established model of cancellous drill hole defect regeneration in sheep, both the proximal and distal epimetaphyseal regions of the femur and the humerus were used bilaterally for eight drill hole cancellous defects (Ø 6 mm, 15 mm depth). Several improvements of the surgical procedure and equipment for an easier harvest of samples were invented. For the inclusion of cortical defect regeneration, a total of eight unicortical diaphyseal drill holes (6 mm Ø) were placed in the proximal-lateral and distal-medial parts of the metacarpal (MC) and metatarsal (MT) diaphyseal bone bilaterally. Acting moments within a normal gait cycle in the musculoskeletal lower limb model were compared with the results of the biomechanical in vitro torsion test until failure to ensure a low accidental fracture risk of utilized bones (ANOVA, p < 0.05). The model was tested in vivo, using thirteen adult, female, black-face sheep (Ø 66 kg; ± 5 kg; age ≥ 2.5 years). In a two-step surgical procedure 16 drill holes were performed for the investigation of two different time points within one animal. Defects were left empty, augmented with autologous cancellous bone or soft bone graft substitutes. Results The in vitro tests confirmed this model a high comparability between drilled MC and MT bones and a high safety margin until fracture. The exclusion of one animal from the in vivo study, due to a spiral fracture of the left MC bone led to a tolerable failure rate of 8 %. Conclusions As a screening tool, promising biomaterials can be tested in this cancellous and cortical bone defect model prior to the application in a more complex treatment site

Can anodised zirconium implants stimulate bone formation? Preliminary study in rat model

Mechanical properties and good biocompatibility of zirconium and some of its alloys focus these materials as good candidates for biomedical applications. The attractive in vivo performance of zirconium is mainly due to the presence of a protective oxide layer. In this preliminary study, surface modification of pure zirconium was made by anodisation in acidic media at low potentials, enhancing the barrier protection given by the oxides and the osseointegration. Electrochemical and SEM (scanning electron microscopy) assays were done in commercially pure zirconium cylinders and two surface conditions were compared: pure and zirconium anodised at 30V. The in vivo assays were held in a tibia rat model. The histological features and fluorochrome labelling changes of newly bone formed around implants were evaluated on the non-decalcified sections 63 days after surgery. Electrochemical and SEM assays showed that anodisation treatment would increased the barrier effect over the material and the in vivo assays showed a continuous newly bone formation around the implant with a different amount of osteocytes in their lacunaes depending the region. There was no significant change in bone thickness around both kinds of implants but conversely a significant increase in the mineralization apposition rate was determined for the anodised condition suggesting that anodisation treatment would stimulate and assist to the osseointegration process. We conclude that anodisation treatment at 30 V can stimulate the implant fixation in a rat model and this study would propose the study of zirconium as candidate material for permanent implant.Fil: Katunar, Maria Rosa. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Mar del Plata. Instituto de Investigación En Ciencia y Tecnología de Materiales (i); Argentina. Universidad Nacional de Mar del Plata. Facultad de Ingeniería; ArgentinaFil: Gomez Sanchez, Andrea Valeria. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Mar del Plata. Instituto de Investigación En Ciencia y Tecnología de Materiales (i); Argentina. Universidad Nacional de Mar del Plata. Facultad de Ingeniería; ArgentinaFil: Ballarre, Josefina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Mar del Plata. Instituto de Investigación En Ciencia y Tecnología de Materiales (i); Argentina. Universidad Nacional de Mar del Plata. Facultad de Ingeniería; ArgentinaFil: Baca, Matías. Hospital Interzonal General de Agudos ‘‘Oscar Alende’’. Mar del Plata; ArgentinaFil: Vottola, Carlos. Hospital Interzonal General de Agudos ‘‘Oscar Alende’’; ArgentinaFil: Orellano, Juan C.. Hospital Interzonal General de Agudos ‘‘Oscar Alende’’. Mar del Plata; ArgentinaFil: Schell, Hanna. Universitatsmedizin Berlin; AlemaniaFil: Duffo, Gustavo Sergio. Comision Nacional de Energia Atomica. Gerencia D/area de Energia Nuclear. Unidad de Actividad de Materiales (cac); Argentina. Universidad Nacional de San Martín; ArgentinaFil: Cere, Silvia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Mar del Plata. Instituto de Investigación En Ciencia y Tecnología de Materiales (i); Argentina. Universidad Nacional de Mar del Plata. Facultad de Ingeniería; Argentin

Early pH changes in musculoskeletal tissues upon injury-aerobic catabolic pathway activity linked to inter-individual differences in local pH

Local pH is stated to acidify after bone fracture. However, the time course and degree of acidification remain unknown. Whether the acidification pattern within a fracture hematoma is applicable to adjacent muscle hematoma or is exclusive to this regenerative tissue has not been studied to date. Thus, in this study, we aimed to unravel the extent and pattern of acidification in vivo during the early phase post musculoskeletal injury. Local pH changes after fracture and muscle trauma were measured simultaneously in two pre-clinical animal models (sheep/rats) immediately after and up to 48 h post injury. The rat fracture hematoma was further analyzed histologically and metabolomically. In vivo pH measurements in bone and muscle hematoma revealed a local acidification in both animal models, yielding mean pH values in rats of 6.69 and 6.89, with pronounced intra- and inter-individual differences. The metabolomic analysis of the hematomas indicated a link between reduction in tricarboxylic acid cycle activity and pH, thus, metabolic activity within the injured tissues could be causative for the different pH values. The significant acidification within the early musculoskeletal hematoma could enable the employment of the pH for novel, sought-after treatments that allow for spatially and temporally controlled drug release

Immune modulation as a therapeutic strategy in bone regeneration

We summarize research approaches and findings on bone healing and regeneration that were presented at a workshop at the 60th annual meeting of the Orthopedic Research Society (ORS) in New Orleans in 2014. The workshop was designed to discuss the role of inflammation in bone regeneration in the context of fundamental biology, and to develop therapeutic strategies that involve immune modulation. Delayed or non-healing of bone is a major clinical problem, with around 10% of fracture patients suffering from unsatisfying healing outcomes. Inflammation is traditionally seen as a defense mechanism, but was recently found essential in supporting and modulating regenerative cascades. In bone healing, macrophages and T- and B-cells interact with progenitor cells, bone forming osteoblasts and remodeling osteoclasts. Among the cells of the innate immunity, macrophages are promising candidates for targets in immune-modulatory interventions that would overcome complications in bone healing and bone-related diseases. Among the cells of the adaptive immune system, CD8+ T cells have been shown to have a negative impact on bone fracture healing outcome, whereas regulatory T cells could be promising candidates that have a positive, modulating effect on bone fracture healing. This workshop addressed recent advances and key challenges in this exciting interdisciplinary research field

T Lymphocytes Influence the Mineralization Process of Bone

Bone is a unique organ able to regenerate itself after injuries. This regeneration requires the local interplay between different biological systems such as inflammation and matrix formation. Structural reconstitution is initiated by an inflammatory response orchestrated by the host immune system. However, the individual role of T cells and B cells in regeneration and their relationship to bone tissue reconstitution remain unknown. Comparing bone and fracture healing in animals with and without mature T and B cells revealed the essential role of these immune cells in determining the tissue mineralization and thus the bone quality. Bone without mature T and B cells is stiffer when compared to wild-type bone thus lacking the elasticity that helps to absorb forces, thus preventing fractures. In-depth analysis showed dysregulations in collagen deposition and osteoblast distribution upon lack of mature T and B cells. These changes in matrix deposition have been correlated with T cells rather than B cells within this study. This work presents, for the first time, a direct link between immune cells and matrix formation during bone healing after fracture. It illustrates specifically the role of T cells in the collagen organization process and the lack thereof in the absence of T cells

Gene identification and analysis of transcripts differentially regulated in fracture healing by EST sequencing in the domestic sheep

BACKGROUND: The sheep is an important model animal for testing novel fracture treatments and other medical applications. Despite these medical uses and the well known economic and cultural importance of the sheep, relatively little research has been performed into sheep genetics, and DNA sequences are available for only a small number of sheep genes. RESULTS: In this work we have sequenced over 47 thousand expressed sequence tags (ESTs) from libraries developed from healing bone in a sheep model of fracture healing. These ESTs were clustered with the previously available 10 thousand sheep ESTs to a total of 19087 contigs with an average length of 603 nucleotides. We used the newly identified sequences to develop RT-PCR assays for 78 sheep genes and measured differential expression during the course of fracture healing between days 7 and 42 postfracture. All genes showed significant shifts at one or more time points. 23 of the genes were differentially expressed between postfracture days 7 and 10, which could reflect an important role for these genes for the initiation of osteogenesis. CONCLUSION: The sequences we have identified in this work are a valuable resource for future studies on musculoskeletal healing and regeneration using sheep and represent an important head-start for genomic sequencing projects for Ovis aries, with partial or complete sequences being made available for over 5,800 previously unsequenced sheep genes

Composite transcriptome assembly of RNA-seq data in a sheep model for delayed bone healing

<p>Abstract</p> <p>Background</p> <p>The sheep is an important model organism for many types of medically relevant research, but molecular genetic experiments in the sheep have been limited by the lack of knowledge about ovine gene sequences.</p> <p>Results</p> <p>Prior to our study, mRNA sequences for only 1,556 partial or complete ovine genes were publicly available. Therefore, we developed a composite <it>de novo </it>transcriptome assembly method for next-generation sequence data to combine known ovine mRNA and EST sequences, mRNA sequences from mouse and cow, and sequences assembled <it>de novo </it>from short read RNA-Seq data into a composite reference transcriptome, and identified transcripts from over 12 thousand previously undescribed ovine genes. Gene expression analysis based on these data revealed substantially different expression profiles in standard versus delayed bone healing in an ovine tibial osteotomy model. Hundreds of transcripts were differentially expressed between standard and delayed healing and between the time points of the standard and delayed healing groups. We used the sheep sequences to design quantitative RT-PCR assays with which we validated the differential expression of 26 genes that had been identified by RNA-seq analysis. A number of clusters of characteristic expression profiles could be identified, some of which showed striking differences between the standard and delayed healing groups. Gene Ontology (GO) analysis showed that the differentially expressed genes were enriched in terms including <it>extracellular matrix</it>, <it>cartilage development</it>, <it>contractile fiber</it>, and <it>chemokine activity</it>.</p> <p>Conclusions</p> <p>Our results provide a first atlas of gene expression profiles and differentially expressed genes in standard and delayed bone healing in a large-animal model and provide a number of clues as to the shifts in gene expression that underlie delayed bone healing. In the course of our study, we identified transcripts of 13,987 ovine genes, including 12,431 genes for which no sequence information was previously available. This information will provide a basis for future molecular research involving the sheep as a model organism.</p