Peter H. Jørgensen

Mechanical properties and biochemical composition of rat cortical femur and tibia after long-term treatment with biosynthetic human growth hormone

The influence of biosynthetic human growth hormone (b-hGH) on female rat cortical femur and tibia was studied after administration of hormone doses of 0.16, 1.10, or 8.33 mg/kg body weight/day for 90 days. The mechanical properties, dimensions, real density, ash weight, and the mineral and collagen concentrations of the bones were measured. In both femur and tibia a positive linear relation was found between the dose of hormone and ultimate load, ultimate stiffness, energy absorption at ultimate load, load at failure, energy absorption at failure, and deflection at failure. In the femur a positive correlation between dose and deflection at ultimate load was also found. After normalizing the mechanical data for the dimensions of the bones, no differences were found in the hormone treated groups compared to placebo, except for the elastic modulus (Youngs modulus), which was decreased in the femur in the group given 8.33 mg b-hGH. The mineral and collagen concentration were unaffected in both femur and tibia, whereas the real density was decreased in the femur. The growth-hormone-induced changes in the mechanical properties seem to be caused mainly by increased dimensions of the bones.

Dose response of growth hormone on fracture healing in the rat.

The effect of different doses of biosynthetic human growth hormone on the mechanical properties of tibial fractures and intact bones was studied in a rat model; a three-point bending test was applied 40 days after fracturing. Ninety-day-old female rats received a daily dose of 0, 0.08, 0.4, 2.0, or 10 mg of growth hormone/kg body weight starting 1 week before fracture and continuing until mechanical testing. In the animals given 2.0 and 10 mg of hormone, the ultimate load sustained by the fractures, stiffness, and energy absorption at ultimate load increased, while the ultimate stress increased only in the latter groups. In the intact bones, ultimate load of the bones increased in the same groups, while stiffness and energy absorption at ultimate load increased only in the group given the highest dose of hormone.

Increased mechanical strength of healing rat tibial fractures treated with biosynthetic human growth hormone.

The effects of biosynthetic human growth hormone on the biomechanical properties of healing tibial fractures and intact bones in the rat were studied after 20 and 40 days of healing. Growth hormone, 2.0 mg per kg per day, was given subcutaneously in two daily doses. Control animals were injected with a corresponding volume of saline. After 20 days of fracture healing, there were no differences in mechanical properties between the healing fractures and intact bones. After 40 days, the ultimate load and maximum stiffness of the fractures of the b-hGH injected animals had increased to more than 400% of the corresponding values of the saline injected animals, and ultimate stress and energy absorption at ultimate load had increased to 270% compared with the saline injected animals. Ultimate load, stiffness, and energy absorption of the intact bones increased in the b-hGH injected animals, but no differences were found in ultimate stress values or normalized energy, indicating that the changes in the intact bones were quantitative phenomena.

Mechanical strength in rat skin incisional wounds treated with fibrin sealant

The biomechanical strength of skin incisional wounds of rats treated with fibrin sealant was assessed by in vitro determination of maximum tensile strength and relative failure energy. Wounds adapted without application of fibrin sealant served as control. Both types of wounds were fixed with surgical tape for the first 8 days of healing. Measurements were performed after 0, 2, 4, 8, 20, and 42 days of healing. After 2 days of healing, wounds treated with fibrin sealant possessed increased maximum tensile strength and relative failure energy. This increase corresponds to the initial strength of the fibrin sealed wounds (0 day values). After 4 and 8 days of healing, no differences were found between the sealed and unsealed groups. After 20 days, the pattern had changed showing increased tensile strength and relative failure energy in wounds not treated with fibrin sealant. A similar trend was reported after 42 days of healing. In both sealed and control wounds, an increase in strain at maximum stress during healing was most pronounced in the first 8 days. After 2 days of healing the strain at maximum stress was increased in wounds treated with fibrin sealant.

Glucocorticoid treatment or food deprivation counteract the stimulating effect of growth hormone on rat cortical bone strength

Growth hormone (GH) has been found to increase the length, thickness and bending strength of rat femora. The present study was designed to investigate if glucocorticoid treatment or food restriction would interfere with the effect of exogenous GH on bone growth. Male rats treated with GH for 30 days experienced a weight gain of 30–35% and longitudinal and periosteal femoral growth. A dose‐related increase in the bending strength of the femora was found and was explained by an increased thickness of the femora. In spite of a reduced real density, biomechanical competence was preserved after GH treatment. GH treatment combined with a relatively small dose of glucocorticoid, which in itself had no significant effect on bone growth and strength, reduced the stimulating effect of GH on body weight gain, femoral growth and strength. GH‐treated rats that were food restricted, so as to limit their body weight gain to that of the saline group, experienced significant longitudinal and periosteal femoral growth. Bone strength, however, was not increased, which conforms to a reduced mineralization and increased porosity of the femora. Youngs modulus (normalized bone stiffness) was significantly decreased in this group, probably as a result of decreased mineralization. Furthermore, the combination of GH treatment and food restriction resulted in a reduced apparent density indicating increased bone resorption.

Growth hormone promotes healing of tibial fractures in the rat.

The effect of administering growth hormone for different periods of time on the mechanical properties of healing rat tibial fractures was investigated after 40 days of healing. Biosynthetic human growth hormone, 2.7 mg/kg body weight/day, was administered to three groups of rats for 1, 2, or 3 weeks following fracture, whereas isotonic saline was administered to a control group for 3 weeks. The ultimate load values and maximum stiffness of the fractures increased in the groups injected with growth hormone for 2 or 3 weeks; linear regression analysis revealed a high probability of a positive linear relationship. In the intact bones an increase in ultimate load, maximum stiffness, and energy absorption at ultimate load was found in the group injected with growth hormone for 3 weeks, with linear regression analysis again showing a high probability of a positive linear relationship.

In situ gentamicin concentrations in cortical bone: An experimental study using microdialysis in bone

We used microdialysis to study gentamicin in cortical bone and compared these results to values obtained in bone specimens. 10 healthy pigs were given an intravenous bolus of 160 mg of gentamicin. We measured reproducibility by inserting two microdialysis catheters into the cortex of tibial diaphyses and by taking samples of serum, dialysates and bone specimens during 6 hours and measuring the concentrations of this antibiotic. The peak concentrations in the two microdialysis probes and bone specimens were 3 mg/L, 2.9 mg/L and 2.6 mg/L (Anova, p= 0.3). Similarly, the areas under the curve from 0 to 6 hours (AUC6h) were 704 mg/min/L, 661 mg/min/L and 569 mg/min/L (Anova, p= 0.07). The reproducibility of the measurements of the microdialysates was evaluated with the mean AUC6h/catheter no.1/AUC6h/ catheter no. 2 ratio, which was 1.12. It seems that microdialysis is a suitable method for making dynamic and quantitative measurements of gentamicin in bone.

Growth hormone increases the biomechanical strength and collagen deposition rate during the early phase of skin wound healing.

The effects of biosynthetic human growth hormone on the biomechanical properties and collagen deposition rate of wound healing were investigated in rat skin incisional wounds after 4 days of healing. Biosynthetic human growth hormone induced a pronounced, ≤94%, and dose‐dependent increase in the mechanical strength of wounds in the dose range of 0.125 to 2 mg/kg/day. A new method for in vivo studies of the collagen deposition rate in granulation tissue of the wound cleft was applied. The production of 3H‐hydroxy‐l‐proline was measured by injecting 3H‐proline intravenously into the rats with a large flooding dose of unlabeled proline which reduces reutilization of 3H‐proline and reduces the influence of de novo synthesis of proline. Extractable collagens, which are not bound in the wound tissue and therefore do not contribute mechanical strength to it, were removed from the samples. Labeled and unlabeled proline were determined simultaneously by reverse‐phase high‐performance liquid chromatography with ultraviolet detection and flow scintillation counting of these amino acids. At day 4 the collagen deposition rate in the incisional wound zone, 0.4 mm wide containing the wound cleft, was 1.8% per hour of the collagen present in the wound zone. The collagen deposition rate was increased by 149% by biosynthetic human growth hormone 2 mg/kg/day compared with the control group. This result indicates that the increased biomechanical strength of the skin incisional wounds of the groups treated with biosynthetic human growth hormone was produced by an increased deposition of collagen in the wound cleft.

Growth hormone, skin and wound healing--experimental studies in the rat.

This thesis is based on experimental works performed at the Department of Connective Tissue Biology, Institute of Anatomy, University of Aarhus, Denmark during my employment as a research fellow. It was a fascinating and most inspiring time and I wish to convey my heartiest thanks to all members of the staff at the institute. Some specific people, however must be mentioned here. Above all special thoughts go to my two friends Associate Professor Troels Torp Andreassen and Associate Professor Hans Oxlund, D.M.Sc.,who introduced me into the fields of collagen and wound healing. With enormous knowledge, patience and perseverance they were ready to discuss and give advice at any time of the day. And they were both able to look far beyond the limited walls of a scientific institute and consider the world in a broader sense. Dr. Bue Bak, D.M.Sc., withwhom I was lucky to share my office was an example to any scientist as he always sticked to the line in the search for his goals. Professor Hans Orskov, D.M.Sc., Institute of Experimental Research, Department of Medicine, Aarhus University Hospital has been a great inspirator due to his enormous knowledge on growth hormone and engagement in any aspect of the exploration of this fascinating molecule. The cooperation with my co-authors Dr. Vet. Karin Damm Jsrgensen, Dr. Christian Bang, Dr. Gitte Ortoft, and Dr. Allan Flyvbjerg, D.M.Sc. has always been a most positive experience for which I am very grateful. Thanks also to the other members of the growth hormone group Dr. Mikkel Seyer-Hansen, Dr. Anne-Marie Briiel, Dr. Hanne Merete Nielsen, Dr. Charlotte Ejersted and Dr. Henrik Christensen for fruitful discussions. Mrs. Eva K. Mikkelsen, Mrs. Lotte Paschburg Kristensen and Mr. Niels Kappel gave me skilled technical assistance and Mrs. Merete Fischer and Mrs. AaseYoung made critical linguistic revisions. Mr. Torkild Alnor Christensen made extraordinary care for the animals. From Novo-Nordisk A/S I deeply thank Anne-Marie Kappelgaard without whose great enthusiasm and open minded attitude the experimental works included in this thesis could not have been performed. Finally, but not less, I want to thank my family my dear wife Birgitte and my children Anders and Kathrine for their love, engagement, encouragement and assistance. The studies were supported by grants from The Danish Health Research Council, The Nordic Insulin Foundation, The NOVO Foundation, NovoNordisk A/S,TheThomas and Elisabeth FrslundNielsen Foundation, The Konsul Johannes FoghNielsen and Fru Ella Nielsen’s Foundation and The Ruth Konig Petersen Foundation. The biosynthetic human growth hormone was kindly donated from the Novo-Nordisk A/S.

Mechanical properties of skin graft wounds.

In female Wistar rats the mechanical strength development of the wound between a skin graft and the neighbouring intact skin (graft wound) was compared with that of ordinary incisional wounds after 4, 7, 14 and 21 days of healing. In one group of rats a 35 x 20 mm skin graft including the subcutaneous muscle was raised and replaced in situ on the left side of the back and a 35 mm incisional wound was made on the right side. In another group a 35 mm incisional wound was made on the right side of the back only. After 4 days the maximum load, maximum stiffness and relative failure energy of the graft wounds were 49, 43 and 40% less respectively than those of the incisional wounds from the same animals and after 7 days the maximum load and maximum stiffness of the graft wounds were reduced by 26 and 29%. However, after 14 and 21 days no differences in mechanical properties were found between these two types of wounds. Compared with the incisional wound from rats without graft the maximum load, maximum stiffness and relative failure energy of the graft wound were reduced by 57, 58 and 44% after 4 days, 59, 62 and 54% after 7 days, 37, 38 and 29% after 14 days and for maximum load and maximum stiffness a reduction of 33 and 31% was found after 21 days of healing.