The effect of aging on bone formation in porous hydroxyapatite: biochemical and histological analysis.

The effect of aging on osteoblastic differentiation of marrow stromal stem cells was examined. Porous hydroxyapatite (HA) disks were soaked in cells suspensions of bone marrow cells from young (8 weeks) and old rats (60 weeks) and then implanted subcutaneously in syngeneic young and old rats. The bone marrow/HA composites were harvested 8 weeks later, and the contents of bone Gla protein (BGP) and alkaline phosphatase (ALP) activity in them were determined. Histologically, bone formation could be detected in all the composites in young recipient rats; however, some old bone marrow/HA composites in old recipients did not show bone formation and the bone volume in the young bone marrow/HA composites was greater than in the old bone marrow/HA composites. The ratios of ALP activities of young bone marrow/HA composites to old bone marrow/HA composites in young and old recipients were about five times and four times, respectively. The ratios of BGP contents of young bone marrow/HA to old bone marrow/HA composite in young and old recipients were about nine and eight times, respectively. The results suggest that the decreased bone formation observed in old bone marrow cells was due to a smaller population of stromal cells and/or decreased capacity of differentiation of stromal stem cells into osteogenic cells.

Osteogenic phenotype expression of allogeneic rat marrow cells in porous hydroxyapatite ceramics.

Porous hydroxyapatite (HA) ceramics were combined with either allogeneic (ACI) or isogeneic (Fischer 344) rat marrow cells and implanted in subcutaneous sites of Fischer rats. FK506 as an immunosuppressant or saline was administered to the recipient rats. The implanted marrow/HA composites were harvested on day 28 and analyzed for bone-forming capability by determining osteoblastic phenotype expression levels of protein synthesis and gene expression. The alkaline phosphatase (ALP) activity and osteocalcin (OC) contents were very low and mRNAs (Northern blot analysis) were not detected in the allografts without FK506. However, high activity of ALP and high content of OC were found and mRNAs were detected in the allografts with FK506 and in the isografts (with and without FK506). This analysis indicates the osteogenic potential of allogeneic marrow cells in the presence of FK506. The histologic sections revealed that allografts without FK506 did not show bone formation but did show the infiltration of many small cells in the ceramics indicating an immunologic reaction, however, the allografts with FK506 and the isografts (with and without FK506) showed consistent de novo bone formation on the HA pore surface. These results indicate that FK506 can suppress the immunologic reaction in the allografts and induce a favorable conditions to support osteoblastic differentiation of allogeneic rat marrow stromal stem cells on the surface of HA ceramics. Therefore, our study suggests the feasibility of clinical transplantation of allogeneic bone marrow for a selected bone graft in applications using adjuvant systemic immunosuppression.

Osteogenic potential of allogeneic rat marrow cells in porous hydroxyapatite ceramics: a histological study.

Hydroxyapatite ceramics facilitate osteogenesis but cannot induce bone formation by themselves. We studied the feasibility of bone formation supported by allogeneic bone marrow cells in porous hydroxyapatite ceramics. Coralline hydroxyapatite discs were soaked in a marrow cell suspension harvested from either ACI (RT1a), Lewis (RT1(1)), or Fischer 344 (RT1(1v)) male rats, and these discs were implanted subcutaneously into 56 male Fischer 344 rats. FK-506 (tacrolimus hydrate), an immunosuppressant, or saline was injected intramuscularly into the recipients every day for 2 weeks after surgery, and additional injections were given to 19 of the rats every 2 days for 2 more weeks. Neither of the mismatched major (ACI rat) or minor (Lewis rat) marrow cell transplants showed any bone formation without administration of FK-506. However, in rats treated with FK-506, bone formed in the pores of all the three types of ceramics implanted, which each contained the marrow cells from one of the three kinds of rats used. There were no differences among the three groups of donors with regard to the bone formation ratio. We previously reported that subcutaneous implantation of porous hydroxyapatite combined with isogeneic marrow cells resulted in consistent bone formation, even at ectopic sites. Since it would be difficult to harvest a large number of autologous marrow cells in clinical cases, we attempted to use allogeneic marrow cells and have shown the allogeneic murine marrow cells to have osteogenic potential.

Vascularized tail bone grafts in rats.

A new experimental model for vascularized corticocancellous bone grafts was established by investigation of vascular anatomy of the tail in 15 adult Fischer 344 rats and determination of the viability of vascularized tail bone grafts into the abdominal wall in 22 7-week-old rats. The tail bones of 40 rats were then raised on the pedicle of the caudal artery and its venae comitantes, transferred to a resected portion in the femur, and observed for 16 weeks. The vascularized graft showed marked reactive periosteal bone formation during the first and second weeks following transfer, and thereafter, the graft continued to show active bone formation. In transverse section, the sharp processes became rounded. In the cancellous bone, both bone resorption and bone formation were noticeably activated early after transfer, although resorption predominated and the amount of the cancellous bone consequently diminished. The nonvascularized grafts showed "creeping substitution." The results suggest that morphologic adaptation occurs if living (i.e., vascularized) tail bones are transferred to long-bone femurs.

Successful replantation of a foot with satisfactory recovery: a case report.

A severed foot in a 51-year-old man was successfully replanted. One year after operation, two-point discrimination was 15 mm on the plantar aspect and 25 mm on the dorsal side. The patient can walk long distances and go up and down stairs without and supportive aids.

Skin graft including subcutaneous vein: experimental study and clinical applications.

Skin grafting, using full and split thickness techniques, may yield poor results when satisfactory circulation is not restored in the recipient bed. Using a rabbit ear model with a vein located in the center of the graft, donor skin and subcutaneous vein were raised simultaneously, and the vein was preserved, resulting in a viable passage for inflow and outflow. Grafts were shown to survive in this experimental group when circulation was satisfactory after venous preservation. In the control group with no vein preservation, all grafts became necrotic. These results were clinically applied for cases in which skin defects, caused by finger trauma, resulted in poor circulation in the recipient bed. Skin and subcutaneous vein were simultaneously taken from the forearm and grafted successfully, resulting in excellent prognoses.

Arteriovenous shunt in digit replantation.

From May 1958 to May 1987, 331 digits were replanted successfully with an overall survival rate of 86.2%. Complete success in replantation of amputated fingers requires an accurate anastomoses of both arteries and veins. However, anastomosis between arteries may not be possible in some patients. Since reports that a thumb amputated at the interphalangeal joint area could be successfully replanted by an arteriovenous shunt on the palmar side, we did arteriovenous shunts in four such cases. Two replantations were successful; necrosis developed in the other two patients.

Pedicled and "flow-through" venous flaps: clinical applications.

Recently, the pedicled venous flap and "flow-through" venous flap have been the focus of increasing attention for skin defects of the fingers and hands. For successful venous flap use, the following approaches have been suggested: (1) a pedicled venous flap with preservation of the draining veins alone; (2) a "flow-through" flap with preservation of a flow-through vein in the flap; and (3) an arterialized "flow-through" venous flap which ensures arterial blood flow into the flap. Based on findings that venous blood is helpful in flap survival, the authors made use of the first two flap types, the pedicled venous flap and the "flow-through" venous flap and attempted to establish and clarify reasonable conditions for flap survival. Venous pressure of the finger and elbow was measured and venographies of the finger and hand were also carried out. The following conditions are regarded as essential in successful venous flap procedures: (a) use of a venous flap with a rich venous network; (b) preservation of many "flow-through" veins; (c) harvesting a pedicled venous flap where the veins have afferent (reversed) venous pressure; and (d) anastomosing veins of the "flow-through" flap with recipient veins where high efferent venous pressure exists and differential pressure is observed. Clinical cases are presented and the authors attempt to explain flap failure from previously unknown causes. Conditions for flap harvesting are also discussed.

Continuous local intra-arterial infusion of anticoagulants for digit replantation and treatment of damaged arteries.

From May 1958 to May 1987, 428 upper extremities were replanted, with an overall survival rate of 87.4 percent. With the aim of increasing the survival rate a new method was tried. Unlike conventional continuous intravenous infusion, a Teflon catheter (28 gauge) was inserted into the proximal main artery of the anastomosed artery, and a daily dose of 80 ml comprising 240,000 U of urokinase, 40 micrograms of prostaglandin E1, 10,000 U (maximum) of heparin, and low molecular weight dextran was administered by means of continuous infusion for the 10 consecutive days, during which arterial thrombosis is likely to occur. Thirteen cases (replantations and damaged digital arteries) survived without any re-operation for arterial thrombus. There were statistically significant differences (p less than 0.025) in platelet count, fibrinogen, and AT III preoperatively and three days postoperatively. There were also statistically significant differences (p less than 0.05) in prothrombin time, partial thromboplastin time, and clotting time between pre-operative measurements and those taken four and 15 hr postoperative, but those data remained within normal range. No abnormalities were present in the arteries through which the catheter was inserted, as validated by postoperative digital subtraction angiography.