New formulations of demineralized bone matrix as a more effective graft alternative in experimental posterolateral lumbar spine arthrodesis.

STUDY DESIGN: A rabbit model of posterolateral intertransverse process spine arthrodesis was used. OBJECTIVE: To determine the efficacy of two new formulations of demineralized bone matrix. SUMMARY OF BACKGROUND DATA: The flowable gel form of Grafton (Osteotech, Eatontown, NJ) demineralized bone matrix has been shown to have osteoinductive properties in various models and currently is used clinically as bone graft material in posterolateral lumbar spine arthrodesis. Two new formulations of Grafton, one made of flexible sheets (Flex) and the other made in a malleable consistency (Putty), have improved handling characteristics compared with the gel form. METHODS: In this study, 108 New Zealand white rabbits underwent bilateral posterolateral intertransverse spine arthrodesis at L5-L6 using autogenous iliac crest bone graft alone (control), one of the new forms of demineralized bone matrix (DBM; made from rabbit bone) alone or in combination with autogenous iliac crest bone. Rabbits were killed 6 weeks after surgery. The lumbar spines were excised, and fusion success or failure was determined by manual palpation and radiography. Specimens also were processed for undecalcified histologic analysis. RESULTS: Manual palpation of the harvested lumbar spines revealed that the fusion rates of the Flex-DBM/Auto group (9/9, 100%) and Putty-DBM/Auto group (10/10, 100%) were superior (P < 0.01) to those of the Auto/control group (3/9, 33%). As a stand-alone graft substitute, Flex-DBM performed superiorly with a fusion rate of 11/11 (100%) compared with that of Putty-DBM (10/12, 83%) and Gel-DBM (7/12, 58%). The devitalized version of Flex-DBM had a fusion rate of 4/11 (36%), which was comparable with the devitalized Putty-DBM rate of 4/12 (33%). Both were superior (P < 0.05) to the devitalized Gel-DBM rate of 0/12 (0%). More mature fusions with greater amounts of trabecular bone were present radiographically and histologically in rabbits that received all forms of demineralized bone matrix than in those in which autograft was used. CONCLUSIONS: The new flexible sheet and malleable putty forms of demineralized bone matrix were effective as graft extender and graft enhancer in a model of posterolateral lumbar spine fusion. These newer formulations of Grafton appear to have a greater capacity to form bone than the gel form or autogenous bone graft alone in this model.

Osteoinduction of human demineralized bone: characterization in a rat model.

The increasing clinical use of human demineralized bone matrix has brought about the desire to understand better the osteoinductivity of these graft materials. The rat heterotopic model has been used successfully to show the sequence of events involved in the endochondral ossification process resulting in osteoinduction. In this study, the osteoinductive potential of human demineralized bone powder was assessed, using immune compromised rats (athymic rnu/rnu) to avoid problems associated with cross species incompatibilities. Implants were placed in subcutaneous or intermuscular sites. This model is characterized to provide a basis for routinely determining the performance of human demineralized bone powder. Demineralized bone powder was prepared from rat and human cortical bone according to a strict protocol. The lack of response to guanidine HCl extracted (noninductive) demineralized bone showed the selectivity of the assay. The same lots of human and rat demineralized bone were tested in sequential experiments during a 1-year period. These results showed reproducible induction of the demineralized bone powder between experiments. Combining demineralized bone with the guanidine HCl extracted demineralized bone in varying ratios tested the sensitivity of the assay. These results showed an increase in bone formation with increasing quantities of active demineralized bone and established the ability of the bioassay to differentiate between the various levels of active (osteoinductive) demineralized bone powder. With this model, consistent performance of demineralized bone powder processed by well controlled methods was seen.

Allograft safety: viral inactivation with bone demineralization.

A study was performed to validate the effectiveness of a bone demineralization process with respect to its inactivation of viruses. The viruses selected for study included human immunodeficiency virus (HIV), duck hepatitis B virus (a model for human hepatitis B), bovine viral diarrheal virus (a model for human hepatitis C), human cytomegalovirus, and human poliovirus (a model for small nonenveloped viruses, e.g., hepatitis A). This study was performed in compliance with Good Laboratory Practice regulations using validation methodology similar to that used to ensure the safety of blood derivatives and other products. Use of the bone demineralization process described in this report resulted in a reduction in infectivity of greater than one million (10(6)) for all viruses and as much as one trillion (10(12)) for the poliovirus.

Current procedures for banking allograft human bone.

The demand for bone and soft tissues for surgical usage has increased rapidly as the efficacy and safety of these materials has been demonstrated. Advances in technology and procedures used to prepare the tissues have also grown in sophistication and remain a dynamic area. How tissue banks screen donors and prepare allografts impacts on the risks and benefits of these materials. Surgeons should understand how tissue banks supplying their graft materials work and have confidence in this treatment option. They will then be prepared to educate their patients to help them make informed decisions concerning allograft usage.

Human safety study of body lotion containing Kathon CG.

The safety of Kathon CG biocide as a preservative in leave-on body lotions was assessed by 2 double-blind studies, using similar protocols. A total of 209 healthy male and female subjects aged 18 to 65 years, 100 in California (72 test subjects, 28 controls) and 109 in Florida (88 test subjects, 21 controls) completed the studies which included pre- and post-use phase diagnostic patch testing with Kathon CG 100 ppm active ingredient, and 13 weeks daily applications of either a test lotion containing Kathon CG 15 ppm active ingredient or a control lotion without Kathon CG. No evidence of irritation or sensitization attributable to use of the biocide was found during regular dermatological examinations during the use phase. Post-use phase patch testing produced negative results in all subjects with the exception of 1 control subject in Florida who had positive readings at the 2- and 4-week post-use phase patch testing. Overall, these studies show there is minimal, if any, risk of adverse effects associated with the use of Kathon CG 15 ppm active ingredient in a leave-on application.

Nifedipine and alpha adrenoceptors in rat aorta. I. Role of extracellular calcium in alpha-1 and alpha-2 adrenoceptor-mediated contraction.

The ability of nifedipine to inhibit contractions induced by non-selective and selective alpha adrenoceptor agonists and by KCl depolarization was studied in the rat aorta. The presence of alpha-2 adrenoceptors which mediate vasoconstriction was demonstrated. Furthermore, this response was highly dependent on extracellular calcium. Alpha-1 adrenoceptors were also present, but this response was dependent primarily upon intracellular calcium stores. Contractions induced by maximally effective concentrations of agonists were separated into fast and slow components of the response. Nifedipine effectively inhibited the slow component of contractions induced by norepinephrine, phenylephrine and clonidine. The slow component of the response induced by these agonists is therefore due to influx of extracellular calcium. The fast component was resistant to nifedipine treatment and is therefore assumed to be due to release of intracellular calcium. Nifedipine was equally potent at inhibiting the slow component of both alpha-1 and alpha-2 adrenoceptor-stimulated contractions and contractions due to KCl depolarization. This suggests that the calcium influx pathways activated by these two types of stimuli 1) may be the same or 2) they have similar affinities for nifedipine or 3) there is a common site of action of nifedipine on both calcium entry pathways.

Nifedipine and alpha adrenoceptors in rat aorta. II. Role of extracellular calcium in enhanced alpha-2 adrenoceptor-mediated contraction in diabetes.

The mechanism responsible for increased norepinephrine-induced responsiveness of aortas isolated from streptozotocin-diabetic rats compared to age-matched control animals was investigated. Selective alpha-1 and alpha-2 adrenoceptor agonists and antagonists were used to determine the contribution of these receptor subtypes to the norepinephrine-induced contractile response. Findings from these experiments indicated an enhancement of alpha-2 adrenoceptor-mediated contraction in aortas from diabetic rats, whereas alpha-1-mediated responses were not altered. The contribution of extracellular calcium influx to the agonist-induced contractions was determined by using the calcium entry blocker nifedipine. Contractile responses to maximally effective concentrations of norepinephrine, phenylephrine and clonidine were separated into fast and slow components and the effect of increasing concentrations of nifedipine on the responses was determined. These experiments indicated that the fast component of contraction to alpha adrenoceptor agonists was insensitive to nifedipine treatment, whereas the slow component was inhibited effectively. The slow component of contraction in response to norepinephrine and clonidine in aortas from diabetic rats was increased significantly compared to control tissues. These results suggest that there is an increase in alpha-2 adrenoceptor activity in aortas from diabetic rats. Furthermore, the increased aortic contractile responses induced by norepinephrine and selective alpha-2 agonists are due probably to an increased influx of extracellular calcium through nifedipine-sensitive ion channels associated with activation of alpha-2 adrenoceptors.

Increased alpha 2-adrenoreceptor mediated vascular contraction in diabetic rats.

Contractile responses of aortic ring preparations from control and diabetic rats to nonselective (noradrenaline) and selective alpha 1-(phenylephrine) and alpha 2-(clonidine) adrenoreceptor agonists were examined to determine the contribution of these receptor subtypes to the response. Aortae from diabetic rats were more responsive to noradrenaline compared to age-matched control rats. There was no difference between contractile responses of aortae from control and diabetic rats to phenylephrine, whereas clonidine-induced contractions were enhanced in aortae from diabetic rats. Dependence of clonidine-induced contractions on extracellular calcium availability was determined. Each of the methods used indicated increased dependency of aortae from diabetic rats on extracellular calcium availability for clonidine-induced contractile force generation. These data indicate that the increased noradrenaline-induced responsiveness of aortae from diabetic rats may be attributed to an increased alpha 2-adrenoreceptor component of the contractile response. It is also concluded that the alpha 2-adrenoreceptor mediated contraction is primarily dependent upon extracellular calcium. From this we suggest that the increased contractile responsiveness of aortae from diabetic rats to alpha 2-adrenoreceptor stimulating drugs may be related to an altered coupling mechanism between the alpha 2-adrenoreceptors and the calcium ion channels or due to a change in membrane permeability as a result of the disease process.