Mechanical stimulation induces pp125(FAK) and pp60(src) activity in an in vivo model of trabecular bone formation.

Utilizing an in vivo model of trabecular bone formation, we demonstrated the temporal and spatial activation of pp125(FAK) in response to specific mechanical load stimuli. Bone chambers equipped with hydraulic actuators were aseptically inserted into each proximal tibial metaphysis of adult, male dogs under general anesthesia. The load stimulus consisted of a trapezoidal waveform, with a maximum compressive load of 17.8 N, loading rate of 89 N/s, at 1 Hz frequency. One chamber was loaded for 2 (120 cycles), 15 (900 cycles), or 30 min (1,800 cycles), whereas the contralateral chamber served as unloaded control. Bone chambers were biopsied at postload time points of 0, 15, and 45 min. Load-induced activation of FAK was rapid, and the duration of activation was dependent on the number of applied load cycles. Mechanical stimulation increased the association of FAK with Src and the time course of complex formation paralleled the temporal activation of FAK. Evaluation of cryosections revealed prominent FAK immunoreactivity among marrow fibroblasts and stromal cells.

An in vivo model for investigations of mechanical signal transduction in trabecular bone.

The premise that bone cells are able to perceive and respond to mechanical forces is well accepted. This article describes the use of an in vivo hydraulic bone chamber for investigations of mechanical signal transduction. The servohydraulic loading mechanism was activated to apply a controlled compressive load to the woven trabecular bone that formed in one chamber, while the contralateral chamber served as an unloaded control. Specimens were harvested at a series of postload time points, and the cellular response to loading was evaluated by cytochemical, histomorphometric, and Northern blot analysis. A repetitive daily load stimulus elicited osteoblast biosynthetic activity characterized by an initial increase in type I procollagen by day 3 and a subsequent rise in alkaline phosphatase (ALP) activity after the sixth daily load episode. Application of a single load episode induced a biphasic pattern of c-fos and zif-268 gene expression with up-regulation at 30 minutes, down-regulation at 12 h, and up-regulation 24 h after the mechanical stimulus. The results show that a synchronized pattern of bone cell activity and gene expression occurs in response to controlled mechanical stimulation and that candidate load-responsive molecular mediators can be evaluated easily by this model.

Perspectives on tissue engineering of bone.

The normal repair and regeneration of bone occurs through an ordered and regulated sequence of cellular events. Successful replacement of bone through tissue engineering likely will be dependent on the recapitulation of this cascade of events. This report presents some of the principles to be considered in the design of engineered bone constructs. The role of cells, a supporting matrix, and endogenous or exogenous biologic or mechanical factors are introduced. The authors' experience with a gene therapeutic approach to bone regeneration is presented as one example of using the principles discussed to promote reproducible bone formation.

Molecular lysis of synovial lining cells by in vivo herpes simplex virus-thymidine kinase gene transfer.

Herpes simples virus thymidine kinase (HSV-TK) expression plasmid DNA was injected into the joint space of rabbits with antigen-induced arthritis (AIA). Purified plasmid DNA was able to mediate transfection of synovial lining cells and transient overexpression of HSV-TK in the context of active synovial inflammation. The pharmacodynamic distribution of intraarticular expression plasmid DNA was confined to the joint space. Arthritic rabbits treated with intraarticular expression plasmid DNA followed by intravenous ganciclovir (GCV, 5 mg/kg) twice daily for 3 days showed histologic evidence of synovial lining layer cytolysis when articular tissues were examined 21 days posttreatment. There was also a reduction in joint swelling in the TK-treated knees. No untoward clinical effects were observed in the rabbits and no evidence of cytolytic damage specific to the TK-GCV gene therapy was observed either in the articular cartilage or bone. The application of TK-GCV intraarticular gene therapy using purified expression plasmid DNA for the induction of synovial cytolysis may be applicable to the treatment of human inflammatory arthritis.

Oxytetracycline-induced nephrotoxicosis in dogs after intravenous administration for experimental bone labeling.

Tetracyclines have been used as in vivo indicators of new bone formation because they form complexes with mineral at bone-forming surfaces. Four of 12 dogs in a bone-labeling study developed clinical signs of renal disease (vomiting, diarrhea, dehydration, and azotemia) within 1 to 2 days of receiving oxytetracycline at a bone-labeling dose of 25 mg/kg of body weight, once daily for 2 consecutive days. To delineate the relationship between oxytetracycline administration and renal damage, six dogs were given the bone-labeling dose intravenously and were subsequently evaluated by determination of clinical signs, serum biochemical analysis, urinalysis, and histologic examination (experiment 1). Drug administration was modified in the five dogs remaining in the bone-labeling orthopedic study. These dogs received the oxytetracycline dose as a slow intravenous infusion diluted with 250 ml of lactated Ringer's solution (experiment 2). All six dogs of experiment 1 developed persistent isosthenuria within 2 days of receiving the bone-labeling dose of oxytetracycline. Clinical illness (three of six dogs) was associated with azotemia, creatinemia, and hyperphosphatemia. All dogs had multifocal, mild to moderate flattening of renal tubular epithelium, characteristic of nephrosis. None of the dogs of experiment 2 developed any clinical indications of renal disease, and the only biochemical abnormality was isosthenuria in two of the five dogs. Thus the development of clinical signs and biochemical abnormalities associated with the intravenous administration of oxytetracycline was obviated by the slow administration of a dilution of the calculated bone-labeling dose of the antibiotic.

Development and use of an animal model for investigations on rotator cuff disease.

Although both intrinsic and extrinsic factors have been implicated in the cause of rotator cuff disease, previous studies have not been designed to test hypotheses of this disease, partly because of the lack of an appropriate animal model. Thirty-three animals were evaluated according to a 34 item checklist of criteria to determine their appropriateness as an animal model for investigations on the rotator cuff. Only the rat shoulder satisfactorily fulfilled all criteria, with a prominent supraspinatus tendon passing under an enclosed arch. Once the rat was identified, 36 rats were randomized to three experimental groups. One group (n = 12) was treated with an intratendinous injection of bacterial collagenase simulating an acute intrinsic injury, another group (n = 12) was treated with an acromial alteration to reduce the subacromial space simulating an external compression, and the third group (n = 12) was treated with a combination of both modifications. Significant increases in cellularity, number of fibroblasts, and collagen disorganization were seen in all experimental tendons compared with a contralateral control group. Semiquantitative grading of histologic sections revealed marked changes in all groups at 4 and 8 weeks. Injuries appeared to be healing at 12 weeks except in the combination group, which seemed to demonstrate persistent alterations. This study supports the rat as an appropriate model for investigating rotator cuff disease. In addition, this study demonstrates that both intrinsic and extrinsic alterations can induce changes in the supraspinatus tendon, which may have similarities to human tendon disease.