Fresh Autologous Marrow Research Articles

The use of bone-marrow-derived fibroblastoid cells and fresh bone marrow in the treatment of bone defects: an experimental study

Bone-marrow aspirate (containing bone progenitor cells), in vitro expanded autologous bone-marrow-derived stromal fibroblastoid cells, and a combination thereof were tested for the potential to fill bone defects. They were compared to grafts of fresh autologous bone or allogeneic devitalized bone. Mandibular defects in rabbits were chosen for this study. The best results were obtained with a combination of in vitro expanded bone-marrow-derived stromal fibroblastoid cells and fresh autologous bone marrow or fresh autologous marrow alone. The effects of these two grafts were similar to grafts of fresh autologous bone and significantly superior to grafts of devitalized allogeneic bone providing only a bone matrix. The in vitro expanded marrow stromal cells induced very significant bone ingrowth, and their effects were only slightly inferior to fresh autologous bone but were superior to devitalized allogeneic bone. These studies suggest that bone marrow is a good source of osteogenic cells both for immediate transplantation and for in vitro expansion and subsequent transplantion.

Characterization of human bone marrow long-term cultures.

In an attempt to more closely simulate the native hematopoietic environment, human bone marrow bony matrix was cultivated in long-term human bone marrow cultures. Sternal bone marrow curettings (i.e., 'bony matrix') were cultured with and without autologous bone marrow single cell suspensions. Fresh media were provided at weekly intervals and the 'harvested' cells were assayed for CFU-gm (i.e., the granulocyte-macrophage precursor). Bone marrow bony matrix alone was not competent to maintain CFU-gm production, although early in the culture nucleated cells and CFU-gm were recruited from the bony matrix. A dose of 850 rad of X-radiation to the bone marrow matrix damaged the hematopoietic growth-promoting effects of the adherent cell layer, which was, however, rapidly reconstituted by an inoculation of intact autologous bone marrow cells. Additional investigations revealed that, whereas the addition of hydrocortisone to this culture system did not alter the maintenance of CFU-gm, recharging the cultures with fresh autologous marrow maintained CFU-gm for 10 weeks. These data indicate that human bone marrow stroma is more sensitive to X-irradiation in vitro than in vivo, and the bone marrow hematopoietic microenvironment provided by its bony matrix seemed to be less effective in vitro than in vivo with respect to the maintenance of hematopoietic precursor cells.

Bridging large bone defects with a xenograft composited with autologous bone marrow. An experimental study.

We have investigated the role of defatted, decalcified xenogenic bone implants composited by impregnation with fresh autologous marrow in bridging large cortical defects. Diaphyseal cortical implants were obtained from orthopaedic operations on humans and from guinea pigs and prepared by treatment with acetone, 0.6 M HCl and ethanol. The composited graft was used to bridge a 3 cm defect in the diaphysis of the ulna of rabbits. Periodic investigations were made clinically, radiographically and histologically and by tetracycline, fluorescence studies up to 20 weeks. Abundant new bone formation was observed in 85.7% of composited human xeno-autografts and in 83.3% of composited guinea pig xeno-autografts. New bone formation was seen in 13.3% of noncomposited xeno-implants. No appreciable antigenicity was observed either clinically or histologically in the grafts. The high degree of success obtained justifies further studies on implants composited by fresh autologous marrow and offers hope for the enhancement of new bone formation clinically, particularly in reconstructive operations for significant skeletal defects. We feel that the osteo-inductive potential of autologous marrow was responsible for the healing of the defects aided by the better internal milieu provided by defatted decalcified xeno implant.

Osteogenesis after bone and bone-marrow transplantation: I. Studies with combined myelo-osseous grafts in the guinea pig

There has been conflicting evidence in the past regarding the contribution made to new bone formation by various components of a bone graft. This study in guinea pigs has compared cortical and cancellous bone allografts, both non-decalcified and hydrochloric acid decalcified, inserted into a muscular site for up to thirteen days, both alone and combined with autologous bone marrow. New bone formation was seen only if the implant contained fresh autologous marrow and it was not seen before the ninth day of implantation. There were no differences in the cell populations about cortical and cancellous grafts. Undecalcified bone evoked giant cell formation as well as new bone.

STUDIES IN THE TRANSPLANTATION OF BONE

1. The present study is an attempt to analyse and apportion significance to the role of inductive mechanisms in bone transplantation. 2. The experimental model used in the present work is that of the composite homograftautograft of cancellous bone previously described (Burwell 1964a). 3. Iliac bone was removed from hooded rats and washed free from its marrow. The bone was then treated by various physical and chemical methods (some of which have been used by other workers to prepare bank bone), namely freezing (-20 degrees Centigrade, -79 degrees Centigrade, -196 degrees Centigrade); freeze-drying (without sterilisation, sterilisation with high energy radiation, sterilisation with ß-propiolactone); decalcification (with E.D.T.A.); irradiation (in the frozen state at a dose of 4 million rads); boiling in water; immersion in merthiolate solution; extraction of organic components with ethylenediamine: and calcining at 660 degrees Centigrade. The treated bone was then impregnated with fresh autologous marrow procured from the femoral shaft of the Wistar rat into which the treated composite graft was to be implanted. The grafts were inserted intramuscularly and removed for study after two, six and twelve weeks. 4. After fixation, serial sectioning and staining, each graft was examined microscopically, and the proportion of new bone/grafted bone scored using an arbitrary scale (0-4). The mean score (and the standard error of the mean score) was then plotted for each treated composite graft and also for several types of fresh cancellous bone grafts. 5. It was found (Fig. 2) that the various treated composite grafts formed a spectrum of bone-forming capacities–the maximum scores being attained by the frozen and freeze-dried composite grafts, the lowest scores by the "deproteinised" composite grafts. 6. The reasons for these differences are discussed. It is concluded that cancellous bone, after transplantation, has the property to induce and promote osteogenesis in marrow; moreover, that this property is contained in the organic components of bone. 7. From the standpoint of inductive mechanisms, cancellous bone treated by freezing or freeze-drying seems to be the most suitable devitalised bone for grafting purposes; bone which has been boiled or merthiolated less suitable; and "deproteinised" bone the least suitable. 8. Freeze-dried bone sterilised physically (by high energy radiation) or chemically (by ß-propiolactone) did not form significantly less new bone than did freeze-dried bone which had not been sterilised. 9. Remodelling mechanisms in bone transplantation are briefly discussed and attention drawn to the deficiencies of present knowledge. The quantitative studies of other workers have indicated that freeze-dried bone may be more rapidly remodelled than is frozen bone. 10. The importance of fresh red marrow in promoting osteogenesis in bone transplantation and in the healing of certain fractures, is emphasised. It seems likely that the interrelationship of bone and marrow revealed by experiment has wider significance not only in health and in response to injury but also in causation of certain idiopathic bone disorders.