Integrins mediate cell adhesion to extracellular matrix components. Integrin alpha 1 beta 1 is a collagen receptor expressed on many mesenchymal cells, but mice deficient in alpha 1 integrin (alpha1-KO) have no gross structural defects. Here, the regeneration of a fractured long bone was studied in alpha1-KO mice. These mice developed significantly less callus tissue than the wild-type (WT) mice, and safranin staining revealed a defect in cartilage formation. The mRNA levels of nine extracellular matrix genes in calluses were evaluated by Northern blotting. During the first 9 days the mRNA levels of cartilage-related genes, including type II collagen, type IX collagen, and type X collagen, were lower in alpha1-KO mice than in WT mice, consistent with the reduced synthesis of cartilaginous matrix appreciated in tissue sections. Histological observations also suggested a diminished number of chondrocytes in the alpha 1-KO callus. Proliferating cell nuclear antigen staining revealed a reduction of mesenchymal progenitors at the callus site. Although, the number of mesenchymal stem cells (MSCs) obtained from WT and alpha 1-KO whole marrow was equal, in cell culture the proliferation rate of the MSCs of alpha 1-KO mice was slower, recapitulating the in vivo observation of reduced callus cell proliferation. The results demonstrate the importance of proper collagen-integrin interaction in fracture healing and suggest that alpha1 integrin plays an essential role in the regulation of MSC proliferation and cartilage production.

Long-bone fracture is a common injury and its healing process at the fracture site involves several overlapping phases, including inflammation, migration of mesenchymal progenitors into the fracture site, endochondral ossification, angiogenesis and finally bone remodelling. Increasing evidence shows that small noncoding RNAs are important regulators of chondrogenesis, osteogenesis and fracture healing. MicroRNAs are small single-stranded, non-coding RNA-molecules intervening in most physiological and biological processes, including fracture healing. Angiogenin-cleaved 5′ tRNA halves, also called as tiRNAs (stress-induced RNAs) have been shown to repress protein translation. In order to gain further understanding on the role of small noncoding RNAs in fracture healing, genome wide expression profiles of tiRNAs, miRNAs and mRNAs were followed up to 14 days after fracture in callus tissue of an in vivo mouse model with closed tibial fracture and, compared to intact bone and articular cartilage at 2 months of age. Total tiRNA expression level in cartilage was only approximately one third of that observed in control D0 bone. In callus tissue, 11 mature 5′end tiRNAs out of 191 tiRNAs were highly expressed, and seven of them were differentially expressed during fracture healing. When comparing the control tissues, 25 miRNAs characteristic to bone and 29 miRNAs characteristic to cartilage tissue homeostasis were identified. Further, a total of 54 out of 806 miRNAs and 5420 out of 18,700 mRNAs were differentially expressed (DE) in callus tissue during fracture healing and, in comparison to control bone. They were associated to gene ontology processes related to mesenchymal tissue development and differentiation. A total of 581 miRNA-mRNA interactions were identified for these 54 DE miRNAs by literature searches in PubMed, thereby linking by Spearman correlation analysis 14 downregulated and 28 upregulated miRNAs to 164 negatively correlating and 168 positively correlating miRNA-mRNA pairs with chondrogenic and osteogenic phases of fracture healing. These data indicated that tiRNAs and miRNAs were differentially expressed in fracture callus tissue, suggesting them important physiological functions during fracture healing. Hence, the data provided by this study may contribute to future clinical applications, such as potential use as biomarkers or as tools in the development of novel therapeutic approaches for fracture healing.

Porous poly(epsilon-caprolactone-co-L-lactide) (P(CL-co-LA, wt % ca. 5/95) sponges were prepared, coated biomimetically with CaP/apatite, and implanted with noncoated control sponges into rat femur cortical defects and dorsal subcutaneous space. The implants were inspected histologically at 2, 4, and 33 weeks after the operation. All implants were filled with fibrovascular tissue within 4 weeks. The femur implants were partially ossified with compact bone, which in the CaP-coated sponges was less mature and more fragmented. Approximately equal amounts of bone were observed in both types of implants. The polymer induced a mild inflammatory reaction with foreign body giant cells but no accumulation of fluid. Degradation of the polymer was slow; most of it was found intact at 33 weeks in histological samples. Nondegraded polymer seems to prevent complete ossification. Cultured osteoblasts proliferated well on apatite-coated material, whereas only a few cells were seen on noncoated material. Thus CaP/apatite coating helped the attachment of osteoblasts in cell cultures but did not offer any advantage in bone formation over noncoated material in vivo. We conclude that a shorter degradation time of P(CL-co-LA) is needed to create an optimal implant. Furthermore, in vivo experiments seem to be necessary for the estimation of osteopromotive properties of a biomaterial.

The fate of intraperitoneally injected or implanted male rat bone marrow-derived stromal cells inside female sibling host animals was traced using Y-chromosome-sensitive PCR. When injected intraperitoneally, Y-chromosome-positive cells were found in all studied organs: heart muscle, lung, thymus, liver, spleen, kidney, skin, and femoral bone marrow with a few exceptions regardless of whether they had gone through osteogenic differentiation or not. In the implant experiments, expanded donor cells were seeded on poly(lactide-co-glycolide) scaffolds and grown under three different conditions (no additives, in osteogenic media for one or two weeks) prior to implantation into corticomedullar femoral defects. Although the impact of osteogenic in vitro cell differentiation on cell migration was more obvious in the implantation experiments than in the intraperitoneal experiments, the donor cells stay alive when injected intraperitoneally or grown in an implant and migrate inside the host. However, when the implants contained bioactive glass, no signs of Y-chromosomal DNA were observed in all studied organs including the implants indicating that the cells had been eliminated.

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(PP) S U M M A R Y Neurofibromatosis type 1 (NF1) is an inherited disease with an incidence of about 1:3000 worldwide. Approximately half of all patients with NF1 present osseous manifestations, which can vary from mild to severely debilitating changes such as congenital pseudarthrosis. In the present study, fracture healing of mouse tibia was followed and specimens were collected 5, 9, 14, and 22 days postoperatively. Experimental pseudarthrosis of rat was followed up to 15 weeks postoperatively. In situ hybridization and immunohistochemistry were used to demonstrate expression of NF1 tumor suppressor and phosphorylated p44/42 mitogen-activated protein kinase (MAPK), an indicator of the Ras-MAPK pathway. The results showed that ossified callus was formed in mouse fracture 22 days after the operation. The final outcome of rat pseudarthrosis was detected 9 weeks after the operation, presenting abundant cartilaginous callus at the pseudarthrosis. NF1 gene expression was noted in the maturing and in the hypertrophic cartilages during normal mouse fracture healing, and in rat pseudarthrosis. Phosphorylated p44/42 MAPK was detected in a subpopulation of the hypertrophic chondrocytes in both models. Furthermore, positive labeling for NF1 mRNA and protein was detected in endothelium in both the pseudarthrosis and in the fracture. In conclusion, NF1 gene expression and function are needed for normal fracture healing, possibly restraining excessive Ras-MAPK pathway activation.

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