Göran Zellin

Importance of delivery systems for growth-stimulatory factors in combination with osteopromotive membranes. An experimental study using rhBMP-2 in rat mandibular defects.

This study was undertaken to investigate whether the choice of carrier/delivery system might be crucial for rhBMP-2 induced osteogenesis beneath osteopromotive membranes. Standardized 5-mm transosseous rat mandibular defects were implanted with recombinant human BMP-2 (rhBMP-2) with or without membrane placement. Two doses of rhBMP-2 (1 microg and 8 microg per defect) were delivered with either collagen sponge or bioabsorbable poly(D,L-lactide-coglycolide) (PLA/PGA) beads plus allogenic blood as carriers. Membrane-covered defects (no BMP) served as controls. Virtually all defects treated with rhBMP-2 without membrane placement already were bridged by new bone after 12 days, independent of rhBMP-2 dose or carrier material, and lateral bone growth was extensive outside the original defect. Membrane placement significantly decreased the stimulatory activity of the BMP, as seen after 12 days, even though osteogenesis was more advanced with rhBMP-2 and membrane compared to membrane alone. After 24 days, defects treated with membrane and rhBMP-2 in the PLA/PGA carrier were totally bridged with regenerated bone, whereas defects covered with membrane without BMP implantation displayed an average bone bridging of only 53%. In an overall analysis of the bone regeneration, the PLA/PGA carrier material was found to be superior to the collagen carrier in the presence of membranes, which was, in turn, more efficient than membrane placement alone (no rhBMP-2). There was much less lateral bone growth when BMP implantation was combined with membrane placement. It was concluded that bone formation beneath osteopromotive membranes may be significantly enhanced by rhBMP-2 and that the delivery system can affect the amount of bone formation obtained. For eventual clinical use, membrane placement has the advantage of keeping the growth-stimulatory implant in place as well as obtaining the desired anatomical contour of the bone formed.

Treatment of Segmental Defects in Long Bones Using Osteopromotive Membranes and Recombinant Human Bone Morphogenetic Protein-2: An Experimental Study in Rabbits

The purpose of this study was to investigate whether barrier membranes that were earlier shown to promote bone healing in the craniofacial skeleton are capable of producing bone healing in long bone. defects by themselves or in combination with recombinant human bone morphogenetic protein 2 (rhBMP-2). Segmental defects (10 mm long) in the rabbit radius, known to heal as pseudoarthrosis-like defects, were used as the experimental model. Treatment with expanded polytetrafluoroethylene membranes (GORE-TEX Membrane) (n = 10) resulted in only minor amounts of bone formation within the defect and collapse of the membranes was common. When placement of membranes was combined with implantation of rhBMP-2 in a beaded biodegradable copolymeric PLA/PGA carrier, total bony bridging of the defects was accomplished within 10 weeks (n = 5). Osteopromotive membranes combined with mBMP-2 can therefore bring about complete healing of long bones. The membranes exclude soft tissue from the defect and at the same time keep the growth-stimulatory implant in place and maintain the anatomical contour of the bone. The combination of osteopromotive membranes and rhBMP-2 may be of. value in reconstructive bone surgery.

Autoclaved bone for craniofacial reconstruction: effects of supplementation with bone marrow or recombinant human fibroblast growth factor-2.

Replantation of resected bone after autoclaving is employed by many units in both craniofacial and orthopedic tumor reconstructive surgery. The procedure is attractive because it maintains the original anatomy, is reliable, and provides a good immediate clinical result. However, doubts have been raised about the ability of the autoclaved bone to revitalize. The present study aimed to explore, in an animal model, the revitalization of autoclaved bone and to determine whether it would be possible to enhance graft revitalization using either autogeneic bone marrow or recombinant human fibroblast growth factor-2, a peptide with stimulatory effects on both endothelial and osteogenic cells. Twenty-eight adult rats were subjected to bilateral parietal cranioplasties (4 x 6 mm), and 75 percent of the grafts were subjected to autoclaving (121 degrees C; 20 minutes) and subsequently treated randomly according to one of the following strategies: no further treatment, or supplementation with bone marrow or recombinant human fibroblast growth factor-2. The remaining grafts were replanted as fresh autografts. The results were evaluated after 4 and 12 weeks by radiologic, histologic, and histomorphometric analyses. After 4 weeks, no major differences were observed between treatments. At 12 weeks, however, no distinction in graft revitalization between autografts and autoclaved grafts supplemented with recombinant human fibroblast growth factor-2 was observed, whereas autoclaved grafts with or without bone marrow displayed significantly less revitalization compared with autografts. The results indicate that autoclaved bone will revitalize by remodeling, and that the efficacy of this process can be increased significantly by simultaneous supplementation with recombinant human fibroblast growth factor-2.

Langerin-expressing and CD83-expressing cells in oral lichen planus lesions

Objective. Dendritic Langerhans cells (LCs) have been attributed a role in the pathogenesis of lichen planus as autoantigen-presenting cells initiating expansion of autoreactive T cells. Langerin and CD83, which are cell molecules expressed on LCs, are associated with antigen presentation. The present study examined expression of Langerin and CD83 molecules on LCs in patients with oral lichen planus (OLP). Material and methods. Biopsies were obtained from seven patients with OLP. Oral mucosa from seven healthy subjects served as controls. Monoclonal antibodies (mAbs) were used in standard immunohistochemical procedures to visualize CD1a-, Langerin-, and CD83-molecule-expressing cells. Results. CD1a+ and Langerin+ cells were found in significantly higher frequencies in OLP epithelium compared with healthy oral epithelium (p<0.01 and p<0.05, respectively); however, the frequency of CD83+ cells did not differ (p>0.05). The connective tissue in OLP lesions showed significantly higher frequencies of CD1a+, Langerin+, and CD83+ cells compared with healthy connective tissue (p<0.01, p<0.01, and p<0.05). CD1a+ and Langerin+ cells in OLP and healthy epithelium had a dendritic morphology. Conclusions. The study shows increased numbers of CD1a- and Langerin-expressing LCs in OLP compared with healthy controls. In the connective tissue, CD83+ cells with dendritic morphology were localized to regions of lymphocyte clusters. The presence of CD83+ dendritic cells in areas of lymphocyte clusters in the connective tissue of OLP lesions indicates the possibility of ongoing autoantigen presentation.

Malformations of the maxillofacial region induced by retinoids in an experimental system

Treatment of pregnant Sprague-Dawley rats with etretinate or retinoic acid on pregnancy Day 8.5-9.0 resulted in craniofacial malformations in 100% of the embryos. A morphological investigation of the maxillofacial malformations was undertaken. Retinoid-exposed embryos showed a reduced skull base, flattened and elongated occiput and micrognathia. The malar bones were reduced or missing. Meckels cartilage was delayed in differentiation as was the mandibular bone. The fusion between different facial processes was disturbed which resulted in facial and palatal clefts. Disturbance of the development of the hypophysis was combined with persisting Rathkes pouch. Aplasia of incisor and molar tooth buds was seen as was aplasia of salivary gland ducts. The facial artery was hyperplastic.

Bone neogenesis in domes made of expanded polytetrafluoroethylene: efficacy of rhBMP-2 to enhance the amount of achievable bone in rats.

For bone reconstructive purposes, it would be a great advantage to be able to gain bone without grafting. In experimental studies, barrier membranes have been used to accomplish this, however, with limited efficacy. In this study, the effect of recombinant human bone morphogenetic protein-2 (rhBMP-2) on the early onset of bone formation, as well as on the final amount of achievable bone, was investigated in an experimental osteo-neogenesis model. In 60 adult rats, dome-shaped barrier membranes made of expanded polytetrafluoroethylene (Gore-Tex membrane), with an inside volume of approximately 60 mm3, were placed on the left parietal bone. The domes were pretreated according to four different alternatives: (1) filled with autogenous blood only (n = 15); (2) filled with 5 microg of rhBMP-2 in an absorbable collagen sponge carrier (n = 15); (3) filled with 15 microg of rhBMP-2 in absorbable collagen sponge carrier (n = 15); or (4) filled with the absorbable collagen sponge carrier only (n = 15). The animals treated according to each alternative were then divided into three equal groups with five rats in each, and subsequently killed after 3, 6, or 12 weeks. The amount of bone formed within the domes was evaluated by light microscopy and computer-assisted image analysis. It was found that the amount of newly formed bone could be enhanced by approximately 100 percent by simultaneous implantation of rhBMP-2, irrespective of dose. The early onset of bone formation was, however, not affected by the rhBMP-2 supplementation. This finding was interpreted as being due to the delivery system used, because as long as the carrier was still present, no significant difference between the treatment groups was observed. The bone formed in domes with carrier implantation, with or without rhBMP-2, displayed more marrow spaces in comparison to controls. The combined treatment with barrier membranes and local delivery of rhBMP-2 may be a useful tool in reconstructive surgery, for instance replacing onlay grafting, especially when a more delicate anatomy is necessary, because membranes can be shaped in multiple ways.

BONE-INDUCTIVE EFFICACY OF RECOMBINANT HUMAN BONE MORPHOGENETIC PROTEIN-2 EXPRESSED IN ESCHERICHIA COLI: AN EXPERIMENTAL STUDY IN RAT MANDIBULAR DEFECTS

Because to our knowledge the efficacy of prokaryotically expressed recombinant human bone morphogenetic proteins (rhBMP) to promote orthotopic osteogenesis has not previously been investigated, our aim was to test the efficacy of rhBMP-2 produced in Escherichia coli to promote bone healing in a standardised experimental bone healing model in rat mandibles. Different doses of rhBMP-2 were delivered in an absorbable collagen sponge carrier, and microporous barrier membranes were placed over half the number of defects in each treatment group, thereby making intraosseous cells the only recruitment source for new osteogenic cells. Results were evaluated by computerised image analysis after 12 and 24 days. The relative efficacy of rhBMP-2 preparations of different purity was also compared. E coli-produced rhBMP-2 stimulated bone healing, but its efficacy was estimated to be about one order of magnitude less than that of rhBMP-2 expressed in eukaryotic cells. We conclude that bacterially expressed rhBMP-2 is osteogenic in vivo, although higher doses will be required than of rhBMP-2 expressed in mammalian cell lines.Because to our knowledge the efficacy of prokaryotically expressed recombinant human bone morphogenetic proteins (rhBMP) to promote orthotopic osteogenesis has not previously been investigated, our aim was to test the efficacy of rhBMP-2 produced in Escherichia coli to promote bone healing in a standardised experimental bone healing model in rat mandibles. Different doses of rhBMP-2 were delivered in an absorbable collagen sponge carrier, and microporous barrier membranes were placed over half the number of defects in each treatment group, thereby making intraosseous cells the only recruitment source for new osteogenic cells. Results were evaluated by computerised image analysis after 12 and 24 days. The relative efficacy of rhBMP-2 preparations of different purity was also compared. E coli-produced rhBMP-2 stimulated bone healing, but its efficacy was estimated to be about one order of magnitude less than that of rhBMP-2 expressed in eukaryotic cells. We conclude that bacterially expressed rhBMP-2 is osteogenic in vivo, although higher doses will be required than of rhBMP-2 expressed in mammalian cell lines.

Characterization of phosphoglycerate mutase isoenzymes from free dissected facial processes

To characterize the three phosphoglycerate mutase (PGM) isoenzymes present in rat facial processes (types MM, BB, and MB), their sensitivity to reagents of the sulfhydryl groups and to heat treatment has been studied. Type BB PGM was not affected by the -SH group reagents; type MB PGM was inhibited about 50%, and type MM PGM was fully inhibited. Type MB PGM showed a greater heat lability than type MM PGM. There was a developmental change from type BB PGM from the 9th embryonic day to isoenzymes MB and MM on the 15th embryonic day. Isoenzyme development was first seen in mandibular processes, followed by maxillary, lateral nasal, and medial nasal processes.

Repeated bone repositioning in the growing rabbit calvarium hampers bone segment incorporation.

&NA; There has been concern among surgeons that multiple extended craniofacial procedures might be detrimental to the viability of the involved skeletal structures. This study aimed to explore the result of repeated bone repositionings in the growing rabbit calvaria. Seven immature rabbits were subjected to a three‐stage surgical procedure, implying that four calvarial bone segments were originally harvested and repositioned according to a rotational scheme; after 6 weeks only three of the bone segments were harvested and repositioned and, finally, after an additional period of 6 weeks, two segments were repositioned. The results were evaluated by radiographic, histologic (with a special scoring system), and histomorphometric analysis. It was found that one or two cranioplasties did not markedly affect graft incorporation, but three procedures significantly reduced graft revascularization and integration. The findings are discussed with special reference to syndromic craniofacial disorders. (Plast. Reconstr. Surg. 100: 619, 1997.)