Longitudinal analysis of osteogenic and angiogenic signaling factors in healing models mimicking atrophic and hypertrophic non-unions in rats.

Impaired bone healing can have devastating consequences for the patient. Clinically relevant animal models are necessary to understand the pathology of impaired bone healing. In this study, two impaired healing models, a hypertrophic and an atrophic non-union, were compared to physiological bone healing in rats. The aim was to provide detailed information about differences in gene expression, vascularization and histology during the healing process. The change from a closed fracture (healing control group) to an open osteotomy (hypertrophy group) led to prolonged healing with reduced mineralized bridging after 42 days. RT-PCR data revealed higher gene expression of most tested osteogenic and angiogenic factors in the hypertrophy group at day 14. After 42 days a significant reduction of gene expression was seen for Bmp4 and Bambi in this group. The inhibition of angiogenesis by Fumagillin (atrophy group) decreased the formation of new blood vessels and led to a non-healing situation with diminished chondrogenesis. RT-PCR results showed an attempt towards overcoming the early perturbance by significant up regulation of the angiogenic regulators Vegfa, Angiopoietin 2 and Fgf1 at day 7 and a further continuous increase of Fgf1, -2 and Angiopoietin 2 over time. However µCT angiograms showed incomplete recovery after 42 days. Furthermore, lower expression values were detected for the Bmps at day 14 and 21. The Bmp antagonists Dan and Twsg1 tended to be higher expressed in the atrophy group at day 42. In conclusion, the investigated animal models are suitable models to mimic human fracture healing complications and can be used for longitudinal studies. Analyzing osteogenic and angiogenic signaling patterns, clear changes in expression were identified between these three healing models, revealing the importance of a coordinated interplay of different factors to allow successful bone healing.

Signaling pathway STAT1 is strongly activated by IFN-ß in the pathogenesis of osteoporosis.

BACKGROUND: Despite extensive research, the underlying pathological mechanisms of osteoporosis are not completely understood. Recent studies have indicated a distinct role for the IFN-ß/STAT1 pathway in bone metabolism. An inhibitory effect of IFN-ß on osteoclastogenesis has been detected and STAT1/2 has been shown to influence osteoblastic bone metabolism. So far, no data concerning the IFN-ß/STAT1 pathways in osteoblasts and osteoclasts from osteoporotic and non-osteoporotic patients are available. The aim of the study was to analyze these pathways in both cell types. METHODS: Osteoblasts were isolated from the femoral heads of 12 osteoporotic and 11 non-osteoporotic patients and monocytes were differentiated into osteoclasts. After the differentiation period, cells were stimulated once with 20 and 100 ng/mL IFN-ß for 4 days. Viability, activity, bone metabolism-related genes, and the proteins Fra1, SOCS1, STAT1, p-STAT1, and TRAF6 were analyzed. RESULTS: Viability, activity, and gene expressions were not affected by stimulating the osteoblasts. However, in osteoporotic osteoclasts, which display a significantly higher basal osteoclastic activity, the stimulation with IFN-ß lead to significant inhibition. Further, an increased STAT1 activation was detected in both cell types with no significant differences between the groups. Regarding the phosphorylation of STAT1, no significant influence was detected in osteoblasts but the IFN-ß stimulation led to a significant increase of p-STAT1 in osteoclasts of both groups. CONCLUSIONS: IFN-ß is a principal mediator in the pathogenesis of osteoporosis by inhibiting osteoclasts and inducing and activating STAT1. Our results also confirm this in cells from osteoporotic and non-osteoporotic patients. Strong inhibitory effects on the osteoclastogenesis of osteoporotic osteoclasts were detectable. Nevertheless, osteoblast activity was not negatively affected by IFN-ß stimulation. These results may contribute to a better understanding of the underlying pathological signaling pathways of osteoporosis.

A pilot study investigating the histology and growth factor content of human non-union tissue.

PURPOSE: The reason for the formation of an atrophic non-union is not clear and an altered vascularization as well as a deregulation of endogenous growth factors is hypothesized. To obtain more information, we analysed human non-union tissue regarding the histology and quantity of several growth factors. METHODS: Tissue from patients with an atrophic non-union (n = 44) or with a healed fracture (n = 13) was analysed. Using histological and immunohistochemical methods the tissue composition was investigated. On the protein level the amount of several growth factors important for bone healing was analysed. RESULTS: The tissue composition was very inhomogeneous containing fibrous, cartilaginous and bony tissue. Vessels were present in all investigated samples without a difference between the tissue from non-union and control patients. The growth factor BMP-2 was below the detection limit in all samples, whereas IL-6 and IGF-I were measured only in a few samples of both groups. TGF-ß1, VEGF-A and BMP-4 were detectable in the majority of the samples of both groups with a high variability in the amount but no difference between the groups. The quantity of both growth factors, BMP-7 and PDGF-AB, was significantly lower in the non-union tissue compared to the healed controls. CONCLUSION: The reduced quantity of BMP-7 and PDGF-AB might be responsible for the impaired healing. Further studies analysing material from more patients and investigating the early healing phases, however, are necessary to obtain further information and consequently improve healing strategies.

Subcutaneous administration of epidermal growth factor: a true treatment option in case of postoperative liver failure?

Adequate hepatocyte regeneration is mandatory for successful recovery after liver resection. The role of epidermal growth factor (EGF), by subcutaneous injection as a simple route, has not been clearly elucidated yet. Wistar rats underwent 70 or 90% hepatectomy, respectively, and were treated with EGF at day 2 and 3 or served as non-EGF-treated controls. Postoperatively, proliferative parameters (weight of liver remnants, number of mitotic and Ki-67 positive cells, expression of cyclin D1 protein) were evaluated. After 90% hepatectomy, 5 day survival was recorded following EGF treatment using different dosages. A significant increase of hepatocellular proliferation was observed after 70% hepatectomy. However, survival following 90% hepatectomy was not as positively affected, irrespective of EGF dosage, with most animals dead before EGF application was completed. Subcutaneous EGF injection can augment postoperative liver regeneration, however, it might be used only as a prophylactic and not as a therapeutic drug in case of liver insufficiency.

Transplantation of monocyte-derived hepatocyte-like cells (NeoHeps) improves survival in a model of acute liver failure.

OBJECTIVE: Investigation of the efficacy of implantation of monocyte-derived hepatocyte-like cells (NeoHeps) in acute liver failure. SUMMARY BACKGROUND DATA: Extended liver resection or split liver transplantation is still associated with high morbidity and mortality because of postoperative liver insufficiency. In view of liver support systems, implantation of isolated hepatocytes or hepatocyte-like cells such as NeoHeps is increasingly under discussion. METHODS: Twenty-four hours before subtotal hepatectomy, cells of different origin [A: human mononuclear cells (24 x 10(6)); B: NeoHeps (16 x 10(6)); C: NeoHeps (24 x 10(6)); D: rat hepatocytes (24 x 10(6))] were injected into the spleen of Wistar rats. After an observation period of 5 days, animal survival, postoperative weight, and signs of encephalopathy were recorded. At the end of the observation period, blood was collected for laboratory analysis. RESULTS: Transplantation of both rat hepatocytes and NeoHeps significantly improved animal survival when compared with control animals (group A: 21%), reaching 72% in group D (P = 0.001), 50% in group C (P = 0.04), and 36% in group B (P = 0.22). Moreover, animals in these groups postoperatively experienced less frequently signs of encephalopathy, as well as earlier weight increase when compared with group A. DISCUSSION: Hepatocyte transplantation is a practicable and successful treatment option in case of liver insufficiency because implantation of NeoHeps or primary rat hepatocytes had an improving effect on survival. The promising data of the present study warrants further analysis to elucidate the role of NeoHeps in treatment of acute postoperative liver failure to a greater extent.