C. R. Howlett

Formation of bone and cartilage by marrow stromal cells in diffusion chambers in vivo.

When freshly isolated rabbit marrow cells were cultured either in vitro or in diffusion chambers in vivo, the hemopoietic cells disappeared and there was a proliferation of the stromal cell population. The colonies formed in vitro were mainly fibroblastic, and this cell type predominated in confluent cultures. Staining for alkaline phosphatase activity and for the Von Kossa reaction was negative in in vitro cultures. However, marrow cell suspensions or fibroblasts harvested from in vitro culture of marrow cells, gave rise to a mixture of bone, cartilage and fibrous tissue in diffusion chambers implanted into the peritoneal cavity. In contrast, only a soft fibrous tissue developed from spleen fibroblasts in diffusion chambers. Differentiation of osteogenic tissue within diffusion chambers fell into two categories: (1) Formation of bone in a fibrous layer surrounding cartilage; (2) intramembranous bone formed directly within fibrous tissue unassociated with cartilage. In both cases alkaline phosphatase activity appeared before the onset of mineralization, and decreased as the first signs of mineral became apparent. The present results suggest that postnatal marrow contains osteogenic precursors with the potential to differentiate via either of the two major paths followed during skeletal development in the embryo. Clonal analysis of the marrow stromal cell population will be required to clarify whether osteo-, chondro-, and fibrogenic cells are the products of one stromal cell line modulated by the microenvironment, or whether there are distinct cell lines for each type.

Fracture healing in a rat osteopenia model.

Fracture healing is influenced by mechanical and biologic factors. The capacity for fracture repair has been reported to decrease with age, although the risk of fracture increases with age. Fracture healing in an animal model of postmenopausal osteoporosis or osteopenia would provide a useful technique to evaluate and develop new treatment protocols. The authors examined the tensile and bending properties of healing femoral fractures in normal and ovariectomized rats. Mechanical data from tensile and bending tests indicate ovariectomy impairs fracture healing, which was confirmed through histologic study. Bending and tensile data provide useful information concerning the mechanical properties of the healing fracture. Tensile tests were noted to be sensitive indicators of the properties of the healing fracture.

Ultrastructure of bone and cartilage formed in vivo in diffusion chambers.

Osteogenic tissue formed in vivo in diffusion chambers inoculated with a suspension of marrow cells contains both bone and cartilage. Observations by transmission electron microscopy on the transitional zone between these tissues showed a mixture of osseous and cartilaginous features in both matrix and cells. Two forms of collagen, morphologically consistent with Types I and II, are found in intimate association within the same intercellular septa. The results suggest the possibility that the different collagen types are synthesized by the same cells and that the variation in cellular morphology could be associated with the changes in the type of collagen synthesized. This unique characteristic of calcified tissues formed in diffusion chambers is probably the result of isolation from direct blood circulation, mechanical stress, and cellular mechanisms of tissue breakdown.

The effect of magnesium ion implantation into alumina upon the adhesion of human bone derived cells

Our group is investigating the potential of modifying the surface atomic layers of biomaterials by ion beam implantation in order to stimulate adhesion of bone cells to these treated biomaterials. In this study alumina that had been implanted with magnesium ions (Mg)−(Al2O3), was compared to unmodified alumina (Al2O3) for the adhesion of cells cultured from explanted human bone. The attachment and spreading of cultured human bone derived cells onto (Mg)−(Al2O3) was significantly enhanced as compared to Al2O3. The role of adsorption of serum adhesive glycoproteins fibronectin (Fn) and vitronectin (Vn) in the adhesion of human bone derived cells to (Mg)−(Al2O3) was determined. The requirement for Fn or Vn for the attachment and spreading of bone-derived cells onto the Al2O3 and (Mg)−(Al2O3) surfaces was directly tested by selective removal of Fn or Vn from the serum prior to addition to the culture medium. The cell adhesion to both the alumina and the (Mg)−(Al2O3) surfaces in the presence of FBS was dependent upon serum Vn.

The inheritance of tibial dyschondroplasia in broilers

A breeding programme to develop a line of chickens with a high incidence of tibial dyschondroplasia (TD) from Australian broiler stock is described. Despite the absence of a control flock, this programme has demonstrated that selection in a broiler population can rapidly increase the incidence of TD. There was consistent circumstantial evidence of the presence of a major sex-linked gene, the recessive allele of which is associated with an increased incidence of TD. The realised heritability estimates (obtained in the absence of a control flock) consistently exceeded 1.00, suggesting the presence of an environmental trend favourable to the onset of TD over the four generations of this investigation. There was a high maternal component or dominance genetic component, or both, for the inheritance of TD in the final generation, suggesting that environmental factors associated with the female parent line may influence the incidence of TD in broilers.

Comparative evaluation of treated bovine pericardium as a xenograft for hernia repair

Two forms of bovine pericardium (BPC) were assessed as hernia repair materials: non-cross-linked (lyophilized) and cross-linked through treatment with glutaraldehyde (GA). These were compared with polypropylene mesh (Marlex) in a rabbit model. Over 52 wk implantation, the GA BPC grafts developed a strong, stable, fibrous tissue replacement with good incorporation into the abdominal muscle wall. The lyophilized BPC grafts were substantially resorbed within 12 wk of implantation, however the thin, fibrous replacement tissue was inadequate for abdominal wall support. Marlex grafts provided sufficient abdominal support, however these grafts were associated with extensive adhesion formation and, in this model, fat deposition around the perimeter of the graft. Control (ungrafted) rabbit abdominal muscle in the transverse orientation had an ultimate tensile load (UTL) of 11.4 +/- 5.1 N (x +/- s.d.) and a strain at UTL of 35 +/- 12% (n = 169). At 52 weeks the UTL of the repair sites was 7.3 +/- 4.5 N (n = 6), 5.1 +/- 3.5 N (n = 6) and 5.6 +/- 2.7 N (n = 6) for GA BPC, lypophilized BPC and Marlex grafts, respectively.

The response of living bone to controlled time-varying loading: method and preliminary results.

Abstract This paper describes a method and equipment for applying controlled sinusoidally-varying axial compressive loading to a typical animal long bone — the ovine metacarpus. The loading is generated hydraulically and applied to the bone through stainless steel bone-pins surgically inserted in the metaphyses. For the ovine metacarpus, the mid-diaphyseal cross-section is well removed from the sites of surgical trauma and from the points of load application. Preliminary results show that the bones responded by adaptive remodelling in the mid-diaphyseal region. The maximum increase in cross-sectional area during a 28-day test period was approximately 17% when the nominal compressive stress was 8.8 MPa. Although a uniform stress field on the mid-diaphyseal region is theoretically possible, it has been found that significant stress non-uniformity may occur due to slight eccentricity of loading.

The functional expression of human bone-derived cells grown on rapidly resorbable calcium phosphate ceramics

The use of biodegradable bone substitutes is advantageous for alveolar ridge augmentation, since it avoids second-site surgery for autograft harvesting. This study examines the effect of novel, rapidly resorbable calcium phosphates on the expression of bone-related genes and proteins by human bone-derived cells (HBDC) and compares this behavior to that of tricalciumphosphate (TCP). Test materials were alpha-TCP, and four materials which were created from beta-Rhenanite and its derivatives: R1-beta-Rhenanite (CaNaPO(4)); R1/M2 composed of CaNaPO(4) and MgNaPO(4); R1+SiO(2) composed of CaNaPO(4) and 9% SiO(2) (wt%); and R17-Ca(2)KNa(PO(4))(2). HBDC were grown on the substrata for 3, 5, 7, 14 and 21 days, counted and probed for various mRNAs and proteins (Type I collagen, osteocalcin, osteopontin, osteonectin, alkaline phosphatase and bone sialoprotein). All substrata supported continuous cellular growth for 21 days. At day 21, surfaces of R1+SiO(2) and R17 had the highest number of HBDC. At 14 and 21 days, cells on R1 and on R1+SiO(2) displayed significantly enhanced expression of all osteogenic proteins. Since all novel calcium phosphates supported cellular proliferation together with expression of bone-related proteins at least as much as TCP, these ceramics can be regarded as potential bone substitutes. R1 and R1+SiO(2) had the most effect on osteoblastic differentiation, thus suggesting that these materials may possess a higher potency to enhance osteogenesis than TCP.

Dupuytren's contracture. Fine structure in relation to aetiology

The fine structure of palmar fascia from patients with Dupuytrens contracture (DC) was compared with that from patients with carpal tunnel syndrome (CTS). In contrast to previous assumptions, the ultrastructure of fibroblasts both in vivo and in vitro from DC and CTS appeared identical, indicating that myofibroblasts are not specific to DC. The major differences between DC and CTS were: 1) a sixfold and fortyfold increase in fibroblast density in cord and nodular areas of DC compared with CTS; 2) a more disorganised pattern of collagen fibrils in DC; and 3) markedly narrowed microvessels surrounded by thickened, laminated basal laminae and proliferating fibroblasts in DC compared with CTS. To account for these morphological changes a hypothesis is presented which proposes that oxygen-free radicals cause pericytic necrosis and fibroblastic proliferation. This hypothesis provides a potential avenue for therapy of DC and other fibrotic conditions.

Autograft and Leeds-Keio reconstructions of the ovine anterior cruciate ligament.

This study compared the Leeds-Keio prosthesis with grafting of autogeneic patellar tendon for the reconstruction of the ovine anterior cruciate ligament under controlled conditions. Reconstructed knees from six sheep of each group were evaluated at 12, 26, and 52 weeks postreconstruction with respect to clinical assessment, gross pathology, mechanical properties, and histology. Although no difference in clinical assessment (anteroposterior draw, range of motion, and function) was noted between the prosthesis reconstruction and the autograft reconstruction, the prosthesis provided a higher strength initially, which remained relatively constant over the one-year study. However, prosthesis wear was observed, with up to 50% of Dacron fibers ruptured in some cases. Histologic sections indicated that in the ovine model, the Leeds-Keio prosthesis should be considered an artificial device and not a scaffold or stent that supports aligned collagenous growth. The autograft had low strength at 12 weeks, which increased over the study period. Despite acceptable clinical performance and adequate mechanical properties up to one year postimplantation, neither reconstruction approached the clinical or mechanical performance of the normal anterior cruciate ligament in the ovine model.