Günter Lauer

The titanium surface texture effects adherence and growth of human gingival keratinocytes and human maxillar osteoblast-like cells in vitro

The adhesion, orientation and proliferation of human gingival epithelial cells and human maxillar osteoblast-like cells in primary and secondary culture were studied on glossy polished, sandblasted and plasma-sprayed titanium surfaces by scanning electron microscopy and in thin sections. The primary cultured explants of human gingival epithelial cells attached, spread and proliferated on all titanium surfaces with the greatest extension on the polished and the smallest extension on plasma-sprayed surfaces. In secondary suspension cultures of gingival keratinocytes, attachment spreading and growth was only observed on polished and plasma-sprayed surfaces, but not on sandblasted surfaces. Moreover, the attachment of these cells depended on the seeding concentration as well as on the coating with fetal calf serum. Cells on polished surfaces developed an extremely flat cell shape, but on sandblasted and plasma-sprayed surfaces a more cuboidal shape. In contrast human maxillar osteoblasts seeded as secondary suspension cultures attached very well to all three differently textured titanium surfaces and showed identical growth patterns independent of the titanium surface structure. These findings suggest that cell morphology, orientation, proliferation and adhesion of human gingival epithelial cells in primary or secondary culture are dependent on the texture of the titanium surface whereas no such differences were observed for maxillar osteoblast-like cells. In conclusion, the soft tissue integration and response is more influenced by the surface texture than the process of osseointegration.

How to optimize seeding and culturing of human osteoblast-like cells on various biomaterials.

The optimization of seeding and culturing of human osteoblast-like cells on three collagen-based biomaterials (bovine, equine and calf collagen membrane) was studied by cell proliferation and cell colonization (scanning electron microscopy) analysis. Osteoblasts of five patients were seeded onto the three biomaterials and two different parameters were varied: the time intervals between initial seeding and adding culture medium (2 h 6 h. 12 h, 24 h) and the seeding concentration (1 x 10(5), 1 x 10(6), 2 x 10(6)cells/ml) of cells onto biomaterials. The results of the study demonstrated that the time interval between seeding osteoblasts and adding culture medium as well as the seeding concentration effects the cell proliferation and the cell colonization. The best proliferation rate was achieved by adding the culture medium 2 h after initial seeding and with a seeding density of 1 x 10(5) cells/ml. Moreover, all three biomaterials resulted in different proliferation rates. The best proliferation rate resulted with the bovine collagen membrane. In conclusion, the examined parameters are very important for the development of the tissue engineering techniques and in a larger perspective also for reconstructive surgery.

Analysis of complications in fractures of the mandibular angle—a study with finite element computation and evaluation of data of 277 patients

AIM The purpose of this study was to compute the load on different osteosynthesis plates in a simplified model using finite element analysis, and to find out whether miniplates were sufficiently stable for application at the mandibular angle. PATIENTS Data from 277 patients with 293 fractures of the mandibular angle have been evaluated. METHODS A computation model using finite elements was established in order to compute mechanical stress occurring in osteosynthesis plates used for fixation of fractures of the mandibular angle. In the second part of this study, the data from all in-patients treated for fracture of the mandibular angle were evaluated retrospectively. Age and sex of the patients, cause of fracture, state of dentition, type of therapy as well as complications were noted. RESULTS In those tests, both the 1.0 mm miniplate and the 2.3 mm module plate were sufficiently stable. The rate of major complications (requiring revisional surgery with general anaesthesia) amounted to approximately 17% in comminuted fractures, or in non-compliant patients in which primary stability with a single miniplate did not appear sufficient, so that other osteosynthesis methods were used in addition. This rate was considerably higher than that in simple mandibular fractures. Simple fractures of the mandibular angle were just treated with one miniplate following Champys guidelines strictly. In these fractures the rate of major complications was only 2.3%. CONCLUSION In comminuted fractures and in non-compliant patients, the use of a stronger osteosynthesis material should be considered while in all other cases application of a single 1.0 mm miniplate was regarded as sufficient for fixation using open reduction.

Prelaminating the fascial radial forearm flap by using tissue-engineered mucosa: improvement of donor and recipient sites.

&NA; In reconstructive surgery, prelamination of free flaps using split‐thickness skin is an established technique to avoid the creation of a considerable defect at the donor site, for example, in the case of a radial forearm flap. For oral and maxillofacial surgery, this technique is less than optimal for the recipient site because the transferred skin is inadequate to form a lining in the oral cavity. To create mucosa‐lined free flaps, prelamination using pieces of split‐thickness mucosa has been performed. However, the availability of donor sites for harvesting mucosa is limited. The present study combines a tissue‐engineering technique with free flap surgery to create mucosa‐lined flaps with the intention of improving the tissue quality at the recipient site and decreasing donor‐site morbidity. On five patients undergoing resection of squamous cell carcinoma of the oral cavity, the radial forearm flap was prelaminated with a tissue‐engineered mucosa graft to reconstruct intraoral defects. Using 10×5 mm biopsies of healthy mucosa, keratinocytes were cultured for 12 days and seeded onto collagen membranes (4.5 × 9 cm). After 3 days, the mucosal keratinocyte collagen membrane was implanted subcutaneously at the left or right lower forearm to prelaminate the fascial radial forearm flap. One week later, resection of the squamous cell carcinoma was performed, and the free fascial radial forearm flap prelaminated with tissue‐engineered mucosa was transplanted into the defect and was microvascularly anastomosed. Resection defects up to a size of 5 × 8 cm were covered. In four patients, the graft healed without complications. In one patient, an abscess developed in the resection cavity without jeopardizing the flap. During the postoperative healing period, the membrane detached and a vulnerable pale‐pink, glassy hyperproliferative wound surface was observed. This surface developed into normal‐appearing healthy mucosa after 3 to 4 weeks. In the postoperative follow‐up period, such functions as mouth opening and closing and speech attested to the success of the tissue‐engineering technique for flap prelamination. (Plast. Reconstr. Surg. 108: 1564, 2001.)

Intraoral wound closure with tissue-engineered mucosa: new perspectives for urethra reconstruction with buccal mucosa grafts.

&NA; In urethra reconstruction, the creation of a new urethra from a free oral mucosa graft is an established surgical technique. The oral mucosa is removed at the same time that the urethra reconstruction procedure is performed. Depending on the size of graft required, the intraoral wound is closed primarily or left to heal secondarily. The latter method limits this technique by leading to scars or strictures, which have a negative impact on the condition of the intraoral soft tissue. Therefore, in this study, a pilot study involving 12 patients, tissue‐engineered mucosa was tested for covering intraoral defects to avoid the drawbacks mentioned above. For mucosa tissue‐graft engineering, a biopsy sample 2 to 4 mm in diameter was removed from the hard palate approximately 4 weeks before the urethra reconstruction procedure was to be performed. In addition, 30 ml of autogenous serum was extracted from a venous whole‐blood sample. The primary cultures were incubated in Dulbecco modified Eagles medium and nutrient factor F 12 (Gibco Co., Eggenstein, Germany), containing the usual additives and autogenous serum. After a period of 3 weeks, subcultivation was performed to engineer mucosa transplants consisting of several layers of keratinocytes on a support foil. After thorough intraoperative blood coagulation had occurred, the cultured mucosa graft on the carrier foil was applied on the wound surface and fixed by single sutures. Additionally, the cultured mucosa graft was covered for 8 to 10 days by an intraoral dressing, which was also fixed onto the wound surface by single suture loops. It is possible to perform primary intraoral wound closure with tissue‐engineered mucosa to cover defect sizes as large as 11.0 × 4.0 cm. This new method provides a better prospect for both urethra reconstruction and the reconstruction of intraoral tissue defects. The number and size of intraoral scars and strictures are diminished. This is of special interest for the reconstruction of the functional unit oral cavity, including soft tissue and cosmetic conditions (e.g., in case of prosthetic rehabilitation). In comparison to primary wound closure with local tissue, the technique presented in this study reduces the severity of postoperative pain and allows faster rehabilitation in patients because of a better woundhealing process. Furthermore, better mobility of intraoral soft tissue structures is achieved. (Plast. Reconstr. Surg. 107: 25, 2001.)

Ectopic bone formation in nude rats using human osteoblasts seeded poly(3)hydroxybutyrate embroidery and hydroxyapatite-collagen tapes constructs

PURPOSE The aim of this study was to evaluate the ectopic bone formation using tissue engineered cell-seeded constructs with two different scaffolds and primary human maxillary osteoblasts in nude rats over an implantation period of up to 96 days. MATERIAL AND METHODS Collagen I-coated Poly(3)hydroxybutyrate (PHB) embroidery and hydroxyapatite (HAP) collagen tapes were seeded with primary human maxillary osteoblasts (hOB) and implanted into athymic rnu/run rats. A total of 72 implants were placed into the back muscles of 18 rats. 24, 48 and 96 days after implantation, histological and histomorphometric analyses were made. The osteoblastic character of the cells was confirmed by immunocytochemistry and RT-PCR for osteocalcin. RESULTS Histological analysis demonstrated that all cell-seeded constructs induced ectopic bone formation after 24, 48 and 96 days of implantation. There was more mineralized tissue in PHB constructs than in HAP-collagen tapes (at day 24; p < 0.05). Bone formation decreased with the increasing length of the implantation period. Osteocalcin expression verified the osteoblastic character of the cell-seeded constructs after implantation time. No bone formation and no osteocalcin expression were found in the control groups. CONCLUSIONS Cell-seeded constructs either with PHB embroidery or HAP-collagen tapes can induce ectopic bone formation. However, the amount of bone formed decreased with increasing length of implantation.

Particle size of hydroxyapatite granules calcified from red algae affects the osteogenic potential of human mesenchymal stem cells in vitro.

Hydroxyapatite (HA) microparticles as a carrier in an injectable tissue-engineered bone filler are considered promising candidates for the treatment of small bone defects in the craniomaxillofacial region. HA granules calcified from red algae, varying in size, were evaluated in vitro for their suitability to be used as a carrier for human mesenchymal stem cells (hMSCs). Three groups of granules were produced in grain sizes of 10–100, 200–500 and 600–1,000 µm. After seeding and culturing hMSCs under osteogenic differentiation conditions onto HA particles for 3, 6 and 9 days, cellular proliferation (tetrazolium salt, XTT), alkaline phosphatase (ALP)-specific activity and total protein synthesis were investigated. The osteoblastic phenotype of the cells was evaluated by assaying the bone-specific genes osteocalcin, osteopontin and collagen type I. XTT assay revealed significantly higher (p < 0.01) proliferation of cells grown on the smallest grain size after 9 days of culture. Regarding ALP-specific activity, significantly higher levels of activity were detected in cells grown on the smallest grain size. Different grain sizes had no significant effects on the secretion of osteocalcin and osteopontin. Collagen type I production was significantly higher (p < 0.05) in cells grown on the biggest grain size in comparison with the two other grain sizes. These results show that the particle size of HA microparticles affects the osteogenic potential of cultured hMSCs and lead to the conclusion that particle size has differential effects on ALP-specific activity and collagen type I production.

Spontaneous tooth eruption after alveolar cleft osteoplasty using tissue-engineered bone: a case report

To bridge the cleft in the alveolar bone and to allow for physiologic eruption of the canine teeth, alveolar bone grafting is often necessary in patients with cleft lips and palates. Instead of autogenous bone, biomaterial seeded with autogenous osteogenic cells has found some clinical application. However, so far no real functional proof has been available to demonstrate that this technique also allows further physiologic features such as tooth eruption to occur. This report describes the results of grafting tissue-engineered bone into the alveolar cleft of a 10-year-old boy. Immediate postoperative healing was uneventful. Eight months after grafting, erupting teeth had moved into the newly formed bone. Eighteen months postoperatively at the site where the tissue-engineered graft had been inserted, the canine had erupted spontaneously in its proper place. The data suggest that tissue-engineered bone can lead to the ossification of the alveolar cleft and allow for physiologic spontaneous tooth eruption.

Tissue-engineered bone grafts for osteoplasty in patients with cleft alveolus

Alveolar bone grafting is an integral part of the treatment concept in cleft palate patients. As an alternative to autogenous bone, tissue-engineered grafts have found some clinical application. The aim of the present study has been to compare ossification in the cleft area using tissue-engineered grafts in a case series of patients with ossification after transplantation of autogenous spongious bone as the gold standard in alveoloplasty. Eight children with complete cleft lips and cleft palates were included in the study. In four children (group A), the cleft defect was filled with tissue-engineered bone (autogenous osteoblasts cultured on demineralized bone matrix Osteovit(®)); as control in another 4 children (group B), the alveoloplasty was performed using spongious iliac bone. Preoperative and 6 months postoperative cone-beam computed tomography was performed, and volumes of the remaining cleft defects were calculated using 3D navigation software. Wound healing was uneventful in both groups. Six months postoperatively the mean volume of the cleft was 0.55±0.24cm(3) after grafting of tissue-engineered bone (group A) and 0.59±0.23cm(3) after transplantation of autogenous spongiosa. In group A, 40.9% of the cleft defect was ossified; in the control group (group B), 36.6%. Tissue-engineered bone is a promising alternative in alveolar bone grafting and no disadvantages were observed in comparison to the gold standard.

Change of mRNA amount of myosin heavy chain in masseter muscle after orthognathic surgery of patients with malocclusion

INTRODUCTION Surgical correction of malocclusion changes the force to moment ratio of masticatory muscles inserting at the mandible caused by shortening, lengthening and rotation of the bone following osteotomy. During muscle adaptation the expression of mRNA for the myosin heavy chain (MyHC) of type I and type II fibres may be changed. MATERIAL AND METHODS The adaptation of the masseter muscle was investigated at the mRNA level in 10 patients 6 months after orthognathic surgery in the mandible. The competitive polymerase chain reaction (cPCR) is a suitable method for quantification of MyHC mRNA. For application of this minimal invasive method an amount of 35 mg muscle tissue was sufficient. RESULTS 6 month postoperatively there was a deficiency of about 87% of MyHC mRNA for fibre type I and II in both groups of patients. The deficiency in patients with mesial position of the mandible was higher but not significant different to patients with distal malocclusion. CONCLUSION Patients should use the postoperative interval for training their masticatory muscles. This improves the stability of treatment result and prevents relapse.