Effect of gamma irradiation on mechanical properties of human cortical bone: influence of different processing methods

The secondary sterilisation by irradiation reduces the risk of infectious disease transmission with tissue allografts. Achieving sterility of bone tissue grafts compromises its biomechanical properties. There are several factors, including dose and temperature of irradiation, as well as processing conditions, that may influence mechanical properties of a bone graft. The purpose of this study was to evaluate the effect of gamma irradiation with doses of 25 or 35?kGy, performed on dry ice or at ambient temperature, on mechanical properties of non-defatted or defatted compact bone grafts. Left and right femurs from six male cadaveric donors aged from 46 to 54?years, were transversely cut into slices of 10?mm height, parallel to the longitudinal axis of the bone. Compact bone rings were assigned to the eight experimental groups according to the different processing method (defatted or non-defatted), as well as gamma irradiation dose (25 or 35?kGy) and temperature conditions of irradiation (ambient temperature or dry ice). Axial compression testing was performed with a material testing machine. Results obtained for elastic and plastic regions of stress-strain curves examined by univariate analysis are described. Based on multivariate analysis it was found that defatting of bone rings had no significant effect on any mechanical parameter studied, whereas irradiation with both doses decreased significantly the ultimate strain and its derivative toughness. The elastic limit and resilience were significantly increased by irradiation with the dose 25?kGy, but not 35?kGy, when the time of irradiation was longer. Additionally, irradiation at ambient temperature decreased maximum load, elastic limit, resilience, and ultimate stress. As strain in the elastic region was not affected, decreased elastic limit resulted in lower resilience. The opposite phenomenon was observed in the plastic region, where in spite of the lower ultimate stress, the toughness was increased due to the increase in the ultimate strain. The results of our study suggest that there may be an association between mechanical properties of bone tissue grafts and the damage process of collagen structure during gamma irradiation. This collagen damage in cortical bone allografts containing water does not depends on the temperature of irradiation or defatting during processing if dose of gamma irradiation does not exceed 35?kGy.     

Albumin-coated structural lyophilized bone allografts: a clinical report of 10 cases

Bone replacement and the use of bone supplementary biological substances have become widespread in clinical practice. Although autografts have excellent properties, their limited availability, difficulties with shaping and donor site morbidity have made allografts a viable and increasingly preferred alternative. The main drawback of allografts is that the preparation destroys osteogenic cells and results in denaturation of osteoinductive proteins. Serum albumin is a well-known constituent of stem cell culture media and we found that lyophilizing albumin onto bone allografts markedly improves stem-cell attachment and bone healing in animal models thus replacing some of the osteoinductive potential. As a first step in the clinical introduction of albumin coated grafts, we aimed to test surgical handling and early incorporation in aseptic revision arthroplasty in humans. We selected patients who needed large structural allografts and the current operation was the last attempt at preserving a moving joint. In a series of 10 cases of hip and knee revision surgery we did not experience any drawbacks of the albumin-coated grafts during handling and implantation. Twelve months radiographic and SPECT-CT follow-up showed that the graft was well received by the host and active remodelling was observed. The lack of graft-related complications and the good 1-year results indicate that controlled trials may be initiated in more common bone grafting indications where long-term effectiveness can be evaluated.     

Solvent-dehydrated calvarial allografts in craniofacial surgery

Craniofacial surgery almost always requires the use of bone grafting. Although autografts are the standard procedure for bone grafting, it is sometimes not possible to harvest bone, and autografts have particular risks. The use of allograft bone provides a reasonable alternative to meet the need for graft material. Solvent dehydration is a multistage procedure in which human cadaveric bone is processed by osmotic exchange baths and gamma sterilization. This processing avoids the risk of infection transmission, decreases antigenicity, and does not weaken the mechanical properties of the bone. Solvent-dehydrated, gamma-irradiated human calvarial bone allografts were used for reconstruction of craniofacial deformities in 24 patients between 1988 and 2002. Resorption of the allografts and results of the surgical intervention were evaluated with plain radiographs and three-dimensional computed tomography 12 months after surgery, in 21 patients. Serologic tests for human immunodeficiency virus-1 antibody, hepatitis B surface antigen, and hepatitis C antigen were also performed. Biopsy specimens were taken from the allografts. Average follow-up in this group was 30 months (range, 8 to 60 months), and results of serologic tests were negative in all patients. Seventy-one percent of the patients (15 of 21) showed no resorption, with partial and complete allograft fusion. One patient had nearly total graft loss and the remaining five patients had 10 to 25 percent graft resorption. Rigid fixation of the allograft, contact with the dura and periosteum, and prevention of dead spaces around the allograft are the most important factors in achieving a satisfactory result. In solvent-dehydrated bone allografts, sterilization and antigenic tissue cleaning are achieved after several steps with a minimal dose of radiation. The result is a nonantigenic, sterile mechanical scaffold that can tolerate external forces. Although autografts are the standard in craniofacial surgery, solvent-dehydrated calvarial bone allografts produced successful results in selected cases.     

Status of Bone Allografting in Japan – Nation-Wide Survey of Bone Grafting Performed from 1995 through 1999

We report the status of bone allografting in Japan on the basis of the information obtained through questionnaires performed by the Japanese Orthopaedic Association (JOA). JOA performed a nation-wide survey in 2000, in order to clarify the current status of musculoskeletal tissue grafting in the orthopaedic practices in Japan. Conducted period was for 5 years from 1995 to 1999. As the results of this survey, it had been clarified that 92,984 bone graftings, which included autografts, allografts and synthetic bone substitutes, were performed during conducted 5 years. While the allografts were used only in 3,212 cases (3%), autograftings were performed in 64,193 cases (69%), synthetic bone substitutes were used in 25,576 cases (28%) in this series. The proportion of the number of operations for use bone substitutes increased every year, whereas that autografting decreased. The proportion of the number of allografting remained almost unaltered. Of the 706 institutions which answered to have experiences of tissue grafting, only 193 (27%) performed allograft.Since Kitasato University Hospital Bone Bank was developed in 1971, we have applied to clinical while doing basic research for preserved bone allograft. When extensive bone graft is required, allograft is very useful. In Japan, however, allograft is not performed widely. The foundation of regional bone banks is expected to resolve this problem. Since excision of bone preparations from cadaver donors is not common, bone allografts are not supplied sufficiently at present. It is needed to develop a network connecting bone banks in Japan. The enlightenment activities to the ordinary people and medical institutions will also be required.     

Prolongation of skin allograft survival after neonatal injection of donor bone marrow and epidermal cells

Composite-tissue (e.g., hand allograft) allotransplantation is currently limited by the need for immunosuppression to prevent graft rejection. Inducing a state of tolerance in the recipient could potentially eliminate the need for immunosuppression but requires reprogramming of the immunological repertoire of the recipient. Skin is the most antigenic tissue in the body and is consistently refractory to tolerance induction regimens using bone marrow transplantation alone. It was hypothesized that tolerance to skin allografts could be induced in rats by injecting epidermal cells with bone marrow cells during the first 24 hours of life of the recipients. Brown Norway rats (RT1n) served as donors for the epidermal cells, bone marrow cells, and skin grafts. Epidermal cells were injected intraperitoneally and bone marrow cells were injected intravenously into Lewis (RT1l) newborn recipient rats. In control groups, recipients received saline solution with no cells (group I, n = 12), bone marrow cells only (group II, n = 15), or epidermal cells only (group III, n = 15). In the experimental group (group IV, n = 18), recipients received epidermal and bone marrow cells simultaneously. Skin grafts were transplanted from Brown Norway (RT1n) rats to the Lewis (RT1l) rats 8 weeks after cell injections. Skin grafts survived an average of 8.5 days in group I (10 grafts), 9.2 days in group II (12 grafts), and 12 days in group III (14 grafts). Grafts survived 15.5 days (8 to 26 days) in group IV (15 grafts). The difference was statistically significant (p < 0.05). Hair growth was observed in some accepted grafts in group IV but never in the control groups. This is the first report of prolonged survival of skin allografts in a rat model after epidermal and bone marrow cell injections. Survival prolongation was achieved across a major immunological barrier, without irradiation, myeloablation, or immunosuppression. It is concluded that the presentation of skin-specific antigens generated a temporary state of tolerance to the skin in the recipients that could have delayed the rejection of skin allografts.     

Comparison of contamination of femoral heads and pre-processed bone chips during hip revision arthroplasty

With bone impaction grafting, cancellous bone chips made from allograft femoral heads are impacted in a bone defect, which introduces an additional source of infection. The potential benefit of the use of pre-processed bone chips was investigated by comparing the bacterial contamination of bone chips prepared intraoperatively with the bacterial contamination of pre-processed bone chips at different stages in the surgical procedure. To investigate baseline contamination of the bone grafts, specimens were collected during 88 procedures before actual use or preparation of the bone chips: in 44 procedures intraoperatively prepared chips were used (Group A) and in the other 44 procedures pre-processed bone chips were used (Group B). In 64 of these procedures (32 using locally prepared bone chips and 32 using pre-processed bone chips) specimens were also collected later in the procedure to investigate contamination after use and preparation of the bone chips. In total, 8 procedures had one or more positive specimen(s) (12.5 %). Contamination rates were not significantly different between bone chips prepared at the operating theatre and pre-processed bone chips. In conclusion, there was no difference in bacterial contamination between bone chips prepared from whole femoral heads in the operating room and pre-processed bone chips, and therefore, both types of bone allografts are comparable with respect to risk of infection.     

Antigen presentation by Langerhans cells in vivo: donor-derived Ia+ Langerhans cells are required for induction of delayed-type hypersensitivity but not for cytotoxic T lymphocyte responses to alloantigens

T cell activation in response to allogeneic stimulation and hapten-specific delayed-contact hypersensitivity responses in vivo can be initiated by Ia-bearing epidermal Langerhans cells (LC). By using a murine heterotopic corneal allograft model, we have investigated the requirement for allogeneic LC as antigen-presenting cells (APC) in the in vivo induction of delayed-type hypersensitivity (DTH) and cytolytic T lymphocyte (CTL) responses to alloantigens in fully allogeneic and H-2 I region-disparate strain combinations. LC-deficient, avascular central corneal allografts from BALB/c donors failed to induce DTH responsiveness when grafted to a subdermal bed on C57BL/6 recipients (p greater than 0.05), yet antigen-specific primary CTL reactivity developed within 7 days after grafting. LC-containing corneal-limbus allografts or central corneal allografts containing a latex bead-induced infiltrate of LC resulted in intense DTH as well as CTL responsiveness when grafted in this same strain combination. Similarly, LC-containing but not LC-deficient corneal allografts from A.TL donors induced DTH responsiveness in I region-disparate A.TH hosts despite the fact that these grafts survived for prolonged duration (less than 28 days). By contrast, CTL induction in I region-disparate hosts was independent of the presence of allogeneic LC. Corneal epithelial cells of grafts removed from I region-disparate hosts 7 days posttransplantation were shown by immunohistology to express the Iak antigens of donor origin. The possibility that bone marrow-derived allogeneic LC were a sufficient requirement for DTH induction was confirmed in experiments performed with CB6F1----B6 bone marrow chimeras used as corneal allograft donors. Corneal-limbus grafts obtained from mice 90 days after chimerization were shown by immunohistology to contain Iad-bearing CB6F1 LC as a sole source of class II alloantigens. When grafted to C57BL/6 recipients, LC-containing chimeric corneas induced DTH responsiveness that was similar in magnitude to that observed in C57BL/6 mice grafted with chimeric skin, yet no DTH response to LC-deficient chimeric central corneal grafts was observed. Moreover, in all cases, the chimeric corneal and skin allografts survived for prolonged duration (greater than 28 days). These results demonstrate that donor-derived LC act as APC in the induction of DTH responsiveness to allogeneic tissue; however, there was no apparent requirement for allogeneic LC in the induction of CTL responses to class I or class II MHC alloantigens.(ABSTRACT TRUNCATED AT 400 WORDS)     

Effect of gamma sterilization on microhardness of the cortical bone tissue of bovine femur in presence of N-Acetyl-L-Cysteine free radical scavenger

Gamma sterilization is usually used to minimize the risk of infection transmission through bone allografts. However, it is believed that gamma irradiation affects the mechanical properties of allografts and free radical scavengers can be used to alleviate the radiation-induced degradation of these properties. The aim of this study was to investigate the radioprotective effects of N-Acetyl-L-Cysteine (NAC) free radical scavenger on the material properties of sterilized bovine cortical bone at microstructure level. Forty-two cortical tissue specimens were excised from three bovine femurs and irradiated to 35 and 70 kGy gamma rays in the presence of 5, 50, and 100 mM concentrations of NAC. The localized variations in microhardness were evaluated via indentation in the radial and longitudinal directions to examine different regions of the microstructures of the specimens, including the osteonal and interstitial tissues. A significant increase was observed in the hardness of osteonal, interstitial, and longitudinal combined microstructures exposed to 35 and 70 kGy radiations (P < 0.05), whereas a relative reduction of the hardness was observed in the radial direction. Furthermore, it was found that the application of 50 and 100 mM NAC during gamma irradiation significantly subsided the hardening in longitudinal combined microstructure. Moreover, the reduction of hardness in radial direction was suppressed in the presence of 100 mM of NAC. In conclusion, the results indicated that NAC free radical scavenger can protect the cortical bone against deteriorative effects of ionizing radiation and can be used to improve the material properties of sterilized allografts.     

Quadriceps tendon allografts as an alternative to Achilles tendon allografts: a biomechanical comparison

Quadriceps tendon with a patellar bone block may be a viable alternative to Achilles tendon for anterior cruciate ligament reconstruction (ACL-R) if it is, at a minimum, a biomechanically equivalent graft. The objective of this study was to directly compare the biomechanical properties of quadriceps tendon and Achilles tendon allografts. Quadriceps and Achilles tendon pairs from nine research-consented donors were tested. All specimens were processed to reduce bioburden and terminally sterilized by gamma irradiation. Specimens were subjected to a three phase uniaxial tension test performed in a custom environmental chamber to maintain the specimens at a physiologic temperature (37 ± 2 °C) and misted with a 0.9 % NaCl solution. There were no statistical differences in seven of eight structural and mechanical between the two tendon types. Quadriceps tendons exhibited a significantly higher displacement at maximum load and significantly lower stiffness than Achilles tendons. The results of this study indicated a biomechanical equivalence of aseptically processed, terminally sterilized quadriceps tendon grafts with bone block to Achilles tendon grafts with bone block. The significantly higher displacement at maximum load, and lower stiffness observed for quadriceps tendons may be related to the failure mode. Achilles tendons had a higher bone avulsion rate than quadriceps tendons (86 % compared to 12 %, respectively). This was likely due to observed differences in bone block density between the two tendon types. This research supports the use of quadriceps tendon allografts in lieu of Achilles tendon allografts for ACL-R.     

Volume maintenance of inlay bone grafts in the craniofacial skeleton

Although the clinical use of inlay bone grafts is widespread in craniofacial surgery, the dynamics of inlay bone grafting to the craniofacial skeleton have never been well characterized. Previous work demonstrated that volume maintenance of bone grafts in the onlay position is a consequence of their microarchitectural features, rather than their embryological origins. The purpose of this study was to investigate whether the properties determining the volume maintenance of bone grafts in the onlay position in the craniofacial skeleton could be extended to bone grafts in the inlay position. It was hypothesized that volume maintenance of an inlay bone graft could be better explained on the basis of the microarchitectural features of the graft (cortical versus cancellous composition), rather than its embryological origin (membranous versus endochondral), and that the primary determinant of bone graft behavior is the interaction between the microarchitectural features of the bone graft and the local mechanical environment in which the bone graft is placed. Cortical and cancellous bone grafts were harvested from the iliac crest (endochondral origin) of 25 New Zealand white rabbits, and cortical bone was harvested from the mandible (membranous origin) of each rabbit. Four 7-mm trephine holes were made in the cranium of each rabbit, posterior to the coronal suture. Each defect was filled with endochondral cortical bone, endochondral cancellous bone, or membranous cortical bone or was left as an ungrafted control specimen. Animals were killed at 3, 8, or 16 weeks. Crania were subjected to micro-computed tomographic and histological assessments. Micro-computed tomographic analysis demonstrated significant increases in actual bone volume from time 0 to the time of death for all types of grafts. Cortical bone demonstrated significant increases in space-occupying volume at all time points. By 16 weeks, no statistically significant difference in either the actual bone volume or the space-occupying volume according to graft type could be detected. There was no resorption of the inlay bone grafts; in fact, all bone types exhibited increased volume. Cancellous bone demonstrated the greatest capacity to increase actual bone volume. All bone graft types seemed to reach a steady-state bone volume, as if controlled by a local regulator. The regulator is likely the local mechanical environment in which the grafts were placed, as corroborated by the findings that the bone grafts seemed to recapitulate the characteristics of the bone in which they were placed, rather than maintaining their native characteristics.     

The effect of heat-killed streptococci on the survival of heart grafts in inbred strains of mice.

Studies with inbred strains of mice revealed that exposure to type 12, Group A, beta-hemolytic streptococci affects the host response to heart grafts implanted in the ear. Intraperitoneal injection of streptococci 10 days before grafting led to curtailed survival of syngrafts without altering the normal rejection time of allografts. Similar sensitization, combined with local injection of streptococci into the graft site at the time of grafting, was followed by rapid rejection of both syngrafts and allografts. The time interval between exposure and grafting was critical. Injection with streptococci 5 days before grafting led to a prolonged survival of allografts and no demonstrable effect on syngrafts. In contrast, injection of streptococci 15 days before grafting did not alter survival of either type of graft. The data indicate heart grafts implanted in the ear may serve as a useful model for the study of the host responses to streptococcal antigens.     

Bone Analysis Using Trabecular Bone Score and Dual-Energy X-Ray Absorptiometry-Based 3-Dimensional Modeling in Postmenopausal Women With Primary Hyperparathyroidism

ObjectivePredominance of bone loss in cortical sites with relative preservation of trabecular bone, even in postmenopausal women, has been described in primary hyperparathyroidism (PHPT). The aim of this study was to evaluate bone microarchitectural differences using dual-energy x-ray absorptiometry (DXA), trabecular bone score (TBS), and DXA-based 3-dimensional (3D) modeling (3D-DXA) between postmenopausal women diagnosed with PHPT (PM-PHPT) and healthy postmenopausal controls.MethodsThis retrospective study included 44 women with PM-PHPT (9 of whom had fractures) and 48 healthy women matched by age, body mass index, and years since menopause treated at Hospital Universitario Fundación Jiménez Díaz between 2008 and 2017. The bone mineral density (BMD) of the lumbar spine (LS), femoral neck, total hip (TH), and 1/3 radius was assessed using DXA, and trabecular volumetric BMD (vBMD), cortical vBMD, integral vBMD, cortical thickness, and cortical surface BMD at TH were assessed using a 3D-DXA software and TBS at LS.ResultsThe mean adjusted BMD values at LS, the femoral neck, and TH; TBS at LS; and TH 3D-DXA parameters (trabecular vBMD, integral vBMD, cortical thickness, and cortical surface BMD) were significantly reduced in women with PM-PHPT compared with those in the controls. However, differences in mean cortical vBMD were not statistically significant (P = .078). There were no significant differences in mean BMD, TBS, or the 3D-DXA parameters between patients with fractures and those without fractures. The 25-hydroxyvitamin D level appeared to be associated with TBS but not with DXA and 3D-DXA measurements.ConclusionPM-PHPT has significant involvement of the trabecular and cortical compartments of the bone, as determined by DXA, TBS, and 3D-DXA.     

The Use of Irradiated Allografts in Reconstruction of Tumor Defects – the Tata Memorial Hospital Experience

A Tissue Bank is a valuable adjunct to tumour management. In bone tumours, the defects produced by ablative surgery can be reconstructed using banked tissue, thereby obviating the donor site morbidity associated with autografts. Allografts are especially useful in large defects or in children where the quantity of available autograft is limited. The use of bone allografts in India has been limited by the availability of good quality, affordable grafts. In this article we share our experience with the use of indigenously produced allografts in limb salvage, as bone graft expanders and as struts. Lyophilised, irradiated bone allografts were morcellised and used in 32 patients. In 21 of these patients the allograft was used in contained cavities. Complete incorporation of the graft was seen between 6-9 months in all the 25 cases available for follow-up. In 4 patients the allograft was layered onto autograft. The allograft incorporated with the host bone in only one of these patients.Struts were used in 9 cases (3 cases complete intercalary segmental defect, 3 cases of hemicortical defects, 2 cases of allograft-prosthesis composite around the hip, 1 case an iliac-crest block was used to stop bleeding from an anterior sacral defect). Of these, no incorporation of the full segment struts was observed in 2 patients who were on chemotherapy and radiotherapy. The sacral defect case was lost to follow-up. All the other struts incorporated with the host bone within 6-9 months. In 5 cases there was sterile postoperative drainage. Overall infection was observed in 4 patients (10%). In one the graft was removed, another settled uneventfully with subsequent incorporation of graft, and two have a persisting sinus but good incorporation. Since radiation and lyophilisation are known to affect the material properties of bone, the grafts were rehydrated in saline for 30 minutes prior to transplantation. Autogenous marrow or autograft was used to provide osteoinductive properties. In selected cases the lyophilised, irradiated bone allografts proved to be clinically useful in the reconstruction of large tumour defects.     

The usefulness of bone metabolic indices for the prediction of changes in bone mineral density after parathyroidectomy in patients with primary hyperparathyroidism.

Patients with primary hyperparathyroidism (pHPT) have reduced bone mineral density (BMD). Although pHPT causes high bone turnover, the exact metabolic bone markers useful for predicting changes in BMD after parathyroidectomy (PTX) remain elusive. The present study was performed to examine the relationship between bone metabolic indices and BMD changes after PTX in 29 pHPT Japanese patients, which received PTX successfully. BMD values were measured by dual-energy X-ray absorptiometry in the lumbar spine and distal one third of radius. As for bone metabolic indices, serum bone-type alkaline phosphates (BAP), serum osteocalcin (OCN), urinary deoxypiridinoline (Dpd), and urinary type I collagen cross-linked N-telopeptides (NTX) were measured. The study included 10 male and 19 female patients (17 postmenopausal). Urinary Dpd, but not NTX was significantly correlated with serum BAP and OCN. Either bone formation or bone resorption indices were significantly and highly correlated with Z-score of BMD in the radius, but not at lumbar spine. Urinary Dpd was significantly correlated with BMD changes at both lumbar spine and radius and at all time points over the two years after PTX. These correlations were most potent among bone metabolic indices in this study. The measurement of urinary Dpd would be useful for predicting long-term changes in BMD at radial and lumbar spine after PTX than other bone metabolic indices.     

Effect of isolation of periosteum and dura on the healing of rabbit calvarial inlay bone grafts

Little is understood about the role of the recipient site in the revascularization and incorporation of autogenous inlay bone grafts in the craniofacial skeleton. Clinical experience demonstrates that secondary complex cranial vault reconstruction performed with scarred avascular dura or poor soft-tissue coverage may undergo significant resorption, thus compromising the aesthetic outcome. This study was designed to determine the effect of isolating autogenous orthotopic inlay calvarial bone grafts from the surrounding dura and/or periosteum on graft revascularization, healing, and volume maintenance in the adult rabbit. Adult rabbits were randomized into four groups (n = 10 per group); in each rabbit, the authors created a circular, 15-mm in diameter, full-thickness cranial defect followed by reconstruction with an autogenous calvarial bone graft, which was replaced orthotopically and held with microplate fixation. Silicone sheeting (0.5 mm thickness) was used to isolate the dura (group II), the periosteum (group II), or both dura and periosteum (group IV) from the graft interface. No silicone was placed in group I. Animals were killed 10 weeks postoperatively, and calvaria were harvested to assess graft surface area, morphology, quantitative histology, fluorochrome staining, and revascularization. Grafts isolated from both the dura and periosteum exhibited significant decreases in total bone (cortical and trabecular) surface area, blood vessel count, and interface healing compared with nonisolated control grafts. Isolation of either the dura or periosteum significantly (p < 0.05) decreased blood vessel count but had no significant effect on interface healing. Isolation of the dura alone was associated with a significant (p < 0.05) decrease in graft cross-sectional surface area and dural cortical thickness compared with nonisolated control grafts, but this effect was not observed when the periosteum alone was isolated. Quantitative histology performed 10 weeks after surgery indicated that graft isolation was associated with increased marrow fibrosis and necrosis compared with nonisolated controls; it also demonstrated evidence of increased activity in bone remodeling (osteoblast and osteocyte count, new trabecular bone, and surface resorption). Triple fluorochrome staining suggested increased bone turnover in the nonisolated grafts compared with isolated grafts at 1 and 5 weeks postoperatively. This study demonstrates that isolating a rabbit calvarial inlay autogenous bone graft from the dura and/or periosteum results in significantly (p < 0.05) decreased revascularization, interface healing, and cross-sectional areas of amount of mature bone compared with nonisolated control grafts 10 weeks after surgery. At this time point, histologic examination demonstrates a paradoxical increase in bone remodeling in isolated bone grafts compared with controls. It is possible that the inhibition of revascularization results in a delayed onset of the remodeling phase of graft incorporation. However, in the model studied, it is not known whether the quantitative histologic and morphometric parameters measured in these isolated grafts exhibit a "catch-up" phenomenon at time points beyond 10 weeks after surgery. The results of this study emphasize the importance of a healthy recipient site in the healing and incorporation of calvarial bone grafts but stress the need for further investigation at later time points.     

Relationship between endogenous parathyroid hormone and bone metabolism/geometry in female patients treated with glucocorticoid.

Although the role of PTH (parathyroid hormone) has been debated in glucocorticoid (GC)-induced osteoporosis (GIO), clinical data about the relation of endogenous PTH to bone metabolism in patients treated with GC are still lacking. The present study was performed to examine the relationship of PTH to bone metabolic indices, bone mineral density (BMD), and bone geometry in 174 female patients treated with oral GC for more than 6 months. Dual-energy X-ray absorptiometry and peripheral quantitative computed tomography (pQCT) were employed for the assessment of BMD and bone geometry. No elevation of serum PTH levels was observed in patients treated with GC. Although serum levels of osteocalcin were not related to serum PTH levels, urinary levels of deoxypiridinoline were positively correlated. Serum PTH levels were negatively related to BMD at any site. In pQCT, serum PTH levels were negatively correlated to both trabecular and cortical volumetric BMD. As for bone morphometric indices, serum PTH levels were significantly related to endocortical circumferences, cortical thickness, and cortical area. Moreover, serum PTH levels were significantly higher in patients with vertebral fractures, compared with those without vertebral fractures in GC-treated patients. In the present study, serum PTH levels were related to the elevation of bone resorption marker, decreased BMD, cortical thinning, and an increase of vertebral fracture risk. The elevation of sensitivity to PTH in bone might play some role in the pathogenesis of GIO.     

A comparative analysis of the microarchitecture of cortical membranous and cortical endochondral onlay bone grafts in the craniofacial skeleton

Previous work in this laboratory established that an onlay bone graft's survival is determined primarily by its relative cortical and cancellous composition rather than its embryologic origin. A volumetric analysis of external bone graft resorption, however, does not explain the internal microarchitectural changes that may be occurring as these grafts become incorporated. To expand the knowledge of bone graft dynamics beyond volumetric parameters, a better understanding of the internal processes of bone graft remodeling is needed. In this comparative study of cortical onlay bone graft microarchitecture, the authors propose to show that cortical onlay bone grafts undergo measurable internal microarchitectural changes as they become incorporated into the surrounding craniofacial skeleton. In addition, the authors propose to further demonstrate similarities between the internal microarchitecture of cortical onlay bone grafts of different embryologic origin over time. Twenty-five adult New Zealand White rabbits were used for this study. They were divided into two groups of eight animals and one group of nine. The groups were killed at 3, 8, and 16 weeks. Cortical membranous and endochondral bone grafts were placed subperiosteally onto each rabbit's cranium. In addition, five ungrafted cortical endochondral and membranous bone specimens were used as controls. Microcomputed tomography (MCT) scanning and histomorphometric analysis were performed on all of the specimens to obtain detailed information regarding the microarchitecture of the cortical bone grafts. The parameters of bone volume fraction, bone surface area to volume, mean trabecular number, and anisotropy were used to give quantitative information about a bone's micro-organization. The results showed that there is no statistically significant difference between the cortical endochondral and the cortical membranous bone grafts for bone volume fraction, bone surface to volume, mean trabecular number, and anisotropy measurements for all time points. There were, however, statistically significant differences when comparing the control and 3-week groups to the 16-week group for all parameters. The advanced MCT technology and histomorphometric techniques proved to be effective in providing a qualitative and quantitative ultrastructural comparison of cortical endochondral and membranous onlay bone grafts over time. In this study, a statistically significant change in the internal microarchitecture of cortical onlay bone grafts of different embryologic origins was seen as they were remodeled and resorbed at all time points. Specifically, the onlay cortical bone grafts developed a less dense, more trabecular, and less organized internal ultrastructure. In addition, no difference in the three-dimensional ultrastructure of cortical endochondral and membranous bone was found. These results challenge some of the currently accepted theories of bone-graft dynamics and may eventually lead to a change in the way clinicians approach bone-graft selection for craniofacial surgery.     

Revascularized periosteal grafts--a new method to produce functional new bone without bone grafting

Rib periosteum was transplanted to the groins of 9 dogs. In half of the periosteal grafts, no microvascular anastomoses were done (free grafts); at 6 weeks after grafting they had become resorbed. The other periosteal grafts were revascularized by microvascular anastomoses of the intercostal vessels to local muscular vessels; at 6 weeks those with confirmed vascular patency had all formed substantial amounts of new bone. Five cm, full-thickness defects were created in the tibias of 10 dogs. The control animals (without grafting) did not heal in two months. However, the experimental dogs, with vascularized periosteal grafts in the defects regenerated their tibias with healthy new bone by 6 weeks--and were walking on them then.     

Very long-term radiographic and bone scan results of frozen autograft and allograft bone grafting in 17 patients (20 grafts) a 30- to 35-year follow-up

In the early 1950s, 48 patients received bone implants from a bone bank in Tel-Hashomer Hospital that stored frozen autograft and allograft bones at temperatures less than -17 degrees C. Seventeen (35%) of these patients (20 implants), 10 men and 7 women, with a mean age of 52.4 (34-69) years were available for follow-up after a mean period of 32.5 (30-35) years. They underwent clinical examination, radiographs and bone scans to evaluate their surgical results. Fracture healing, non-union, graft resorption, osteoporosis and bone sclerosis were used as radiographic criteria for bone incorporation, and normal, increased and decreased uptake served to assess the bone scan. Based on the above criteria, the results were satisfactory in 17 (85%) and poor in 3 (15%). The three failures were after shelf operation for hip dysplasia that used two allografts and one autograft. Allogenous or a combination of allogenous with autogenous frozen bone grafts proved to be a satisfactory and durable method for filling bone defects.     

Sporicidal efficacy of genipin: a potential theoretical alternative for biomaterial and tissue graft sterilization

Terminal sterilization of musculoskeletal allografts by gamma radiation minimizes the risk of disease transmission but impairs allograft mechanical properties. Commonly employed crosslinking agents can sterilize tissues without affecting mechanical properties adversely; however, these agents are toxic. Genipin is reported to be a benign crosslinking agent that strengthens mechanical properties of tissues; however, the antimicrobial capacity of genipin is largely unknown. The present study’s aims were: (1) to assess the sporicidal potential of genipin, (2) to improve antimicrobial capacity by changing chemical and physical treatment conditions. To establish genipin’s sterilization potential Bacillus subtilis var. niger spore strips were treated with 0–10 % genipin in PBS or in 1:1 DMSO:PBS up to 72 h at room temperature (RT). Sterilizing doses and concentrations of genipin were used to treat B. pumilus and Geobacillus stearothermophilus spores to assess broader spectrum sporicidal activity of genipin. Scanning electron microscopy (SEM) was performed to evaluate gross morphological changes after genipin treatment. Optimal sterilization conditions were determined by evaluating the effects of temperature (RT-50 °C), DMSO:PBS ratio (0:100–100:0), and treatment duration (24–72 h) on B. subtilis. Genipin penetration of full thickness bovine patellar tendon and cortical bone specimens was observed to assess the feasibility of the agent for treating grafts. Initial studies showed that after 72 h of treatment at RT with 0.63–10 % genipin/DMSO:PBS B. subtilis spore strips were sterilized; 0.63 % genipin/PBS did not sterilize spore strips at 72 h at RT. Genipin doses and concentrations that sterilized B. subtilis spore strips sterilized B. pumilus and G. stearothermophilus spore strips. SEM revealed no gross morphological differences between untreated and treated spores. Treatment optimization resulted in sterilization within 24 h with 100 % PBS, and DMSO facilitated sporicidal activity. Genipin penetrated full thickness patellar tendon specimens and 3.72 ± 0.58 mm in cortical bone specimens. Genipin sterilizes B. subtilis, B. pumilus, and G. stearothermophilus spore strips. It penetrates soft and hard tissues at doses previously shown to be non-toxic and to improve mechanical strength in collagen-rich soft tissues. Further studies are indicated to assess genipin’s effects on the mechanical properties of genipin-sterilized grafts, the ability of genipin to eradicate infectious species other than spores, and to assess whether sterilant activity persists after penetrating tissues and biomaterials.