Primary orbital reconstruction with selective laser melted core patient-specific implants: overview of 100 patients.

Contemporary advances in technology have enabled the transfer of industrial laser melting technology to surgery, and its use can improve the accuracy of orbital restoration. The aim of this study therefore was to evaluate the accuracy of primary orbital reconstruction with the use of selective laser melted, patient-specific implants and navigation. A total of 100 patients with complex orbital fractures were included. Planned orbital volumes were compared with those achieved, and angles were compared with the unaffected side. Analysis included the overlay of postoperative on planned images (iPlan® 3.0.5, Brainlab). The mean (SD) orbital volume of the unaffected side was 27.2 (2.8)ml in men and 25.0 (2.6)ml in women. Fractures that involved the posterior third of the orbital floor and comminuted fractures showed significant orbital enlargement (p=0.026). The mean (SD) reconstructed orbital volume was 26.9 (2.7)ml in men and 24.26 (2.5)ml in women. Three-dimensional analysis of the colour mapping showed minor deviations when compared with the unaffected side. The results suggest that a high degree of accuracy can be routinely achieved in these complex cases.

Evaluation of the quality of life of patients with maxillofacial defects after prosthodontic therapy with obturator prostheses.

The purpose of this study was to investigate how patients with maxillofacial defects evaluate their quality of life after maxillectomy and prosthodontic therapy with obturator prostheses. 43 patients were included in the study (25 female, 18 male). 31 (72%) patients completed a standardized questionnaire of 143 items and then answered additional questions in a standardized interview. Global quality of life after prosthodontic therapy with obturator prostheses was 64% (±22.9) on average. Functioning of the obturator prosthesis, impairment of ingestion, speech and appearance, the extent of therapy, and the existence of pain had significant impact on the quality of life (p<0.005). Orofacial rehabilitation of patients with maxillofacial defects using obturator prostheses is an appropriate treatment modality. To improve the situation of patients prior to and after maxillectomy sufficient information about the treatment, adequate psychological care and speech therapy should be provided.

The new polyomavirus (MCPyV) does not affect the clinical course in MCCs.

Since 2008, a new polyomavirus (MCPyV) in Merkel cell carcinomas (MCC) has been described, but little is known about its impact on the clinical course. The purpose of this study was to determine the presence of MCPyV in a large sample and to correlate the results with the clinical course of the disease. 59 samples from 44 patients were analysed for the presence of MCPyV using the primers LT3, VP1 and LT1. The clinical records of these patients were evaluated and correlated with the presence of MCPyV. 58% of specimens were positive for MCPyV. Of these, LT3 was positive in 53%, VP1 in 37% and LT1 in 10%. 57% of primary tumours and 53% of metastases were positive for LT3; the numbers for VP1 and LT1 were lower. There was no correlation between the detection of MCPyV in the primary tumour and the appearance of metastases. The survival time was statistically independent from the presence of MCPyV. There is a striking occurrence of MCPyV in MCC, but whether it affects the clinical course remains unclear.

[On quality of life after surgical therapy of oral cancer - a retrospective multi-center study: the connection between dedentition, denture, quality of life, and dysphagia, and the resulting rehabilitation schemes]. / Zur Lebensqualität nach chirurgischer Therapie von Mundhöhlenkarzinomen - eine retrospektive Multicenterstudie: Der Zusammenhang zwischen Zahnverlust, Zahnersatz, Lebensqualität und Dysphagie sowie die daraus resultierenden Massnahmen bei der Rehabilitation.

PURPOSE: The surgical treatment of oral cancer results in functional and aesthetical impairments. Patients' quality of life is considerably impaired by oral symptoms resulting from therapy of oral cancer. In many cases the inevitable resection of the tumor, as well as the adjuvant radiochemotherapy will cause the destruction of physiologically and anatomically important structures. One focus of research was the specific rehabilitation of dental loss by functional dentures. Another was the course of 19 impairments (comprehension of speech for unknown others, comprehension of speech for familiar others, eating/swallowing, mobility of the tongue, opening range of the mouth, mobility of lower jaw, mobility of neck, mobility of arms and shoulders, sense of taste, sense of smell, appearance, strength, appetite, respiration, pain, swelling, xerostomia, halitosis). METHODS: Commissioned by the German, Austrian and Swiss cooperative group on tumors of the maxillofacial region (DOSAK), data were collected in 3.894 questionnaires at 43 hospitals in Germany, Austria and Switzerland. The catalogue comprised 147 items in 9 chapters. At the end of the enquiry, 1.761 anonymous questionnaires were returned by 38 hospitals. 1.652 of these could be evaluated regarding the question. RESULTS: The sum score of the 19 impairments was highly increased immediately after the operation and recovered over the next 6 months, without, however, reaching the pre-surgery level. Of 1.652 patients, only 35% did not lose any teeth during therapy. 23% lost up to 5, 17% up to 10 teeth. A quarter of the patients lost more than 10 teeth. The more teeth were lost, the greater the decline of quality of life (p < or = 0.001), although this could be allayed by the functionality of the dentures (p < or = 0.001). There is a reciprocal dependence between the functionality of dental prosthetics and impairment by eating/swallowing (p < or = 0.001). CONCLUSIONS: Patients' quality of life after radical surgery of a carcinoma of the oral cavity depends not only on the functionality of dentures and the specificity of rehabilitation, but also from the initial findings, the extent and location of the resection, the chosen therapy, the general circumstances of the patient's life as well as their strategies of coping. These factors, however, unlike those of functionality of dental prosthesis and rehabilitation, are not modifiable.

[Causation, therapy and complications of treating mandibular fractures - a retrospective analysis of 10 years]. / Ursachen, Therapie und Komplikationen bei der Frakturversorgung des Unterkiefers - eine retrospektive Analyse von 10 Jahren.

BACKGROUND: Fractures of the mandible are a common form of facial injury. The aetiological factors associated with mandibular fractures and the trends in these factors over a 10-year period are reported. METHODS: A retrospective survey was carried out of 724 patients presenting with a fracture of the mandible over the 10-year-period 1994-2003. Patients; records were reviewed and analyzed according to age, sex, cause of injury, anatomic site of fracture, treatment and postoperative complications. RESULTS: Over the 10-year-period the rate of mandibular fractures remained constant (mean 40,7%). There were no changes in the age group (mean 33,3 years) or in the higher prevalence in male (male-female-ratio 2,3 : 1). The major causes of fractures were assaults (38,6%) and accidental fall (27,3%). The most common fracture site was the condylar region (47,0%) followed by the angle (29,4%). Most fractures were treated by closed reduction until 2002, thereafter surgical treatment increased noticeable. The complication rate was 8,9% and the most common complications were hardware exposure and infection. CONCLUSION: Fractures of the mandible are a prevalent form of facial injury. Aetiological factors show no significant change over the 10-year-period. Complication-rate is low and will support the tendency towards surgical treatment.

A prospective comparison of octyl-2-cyanoacrylate and suture in standardized facial wounds.

Regarding the cosmetic results of wound closure using tissue adhesives as opposed to sutured wounds, most published studies are performed on children and with non-standardized lacerations, which makes it difficult to transfer the results to facial incisional wounds in adults. There are also conflicting conclusions about the cosmetic results. The purpose of this randomized prospective study was to compare the cosmetic outcomes of wound closure with sutures and octyl-2-cyanoacrylate in standardized facial wounds in adults. To compare very similar facial wounds, the infraorbital cut (lower eyelid incision) was used in 45 patients. The assessment was performed by patients and surgeons blind to the data. Phase-shifting profilometry was used to measure the profile of the scars. No early complications were observed in any group. The wound depth in the tissue adhesive group was significantly greater than in the suture group. There were no differences in the overall cosmetic results among all patients in the two groups. Interestingly, in the younger patients the result of the sutured wounds was superior to that of the adhesive-treated patients. The Dermabond skin adhesive provides a means of closure of facial surgical wounds without early complications, but adjustment of the edges of the wound and the cosmetic result in younger patients are less successful than with thin sutures.

[Medical expert opinions in oral and maxillofacial medicine regarding incisor tooth trauma]. / Frontzahntraumata als Gegenstand von Gutachten.

BACKGROUND: The treatment of traumatized patients plays a significant role in the everyday life of oral and maxillofacial surgeons as well as of dentists. Traumas of incisor teeth account for a considerable part. These traumas are often followed by lawsuits. The aim of this retrospective analysis was to highlight aspects of these lawsuits of special significance and to show the consequences for surgeons and general practitioners. MATERIAL AND METHODS: A total of 398 medical expert opinions were evaluated. Moreover, an online search of databases for relevant court decisions was performed. Analysis was initially based on formal aspects of the cases and reports. It was later supplemented by a differentiated assignment of the questions addressed by the courts to the expert consultants. RESULTS: In 97 (24%) medical expert opinions traumas of the incisor teeth were the main subject. In 35% the trauma was caused by traffic accidents followed by assaults. In one-third the medical expert opinion was commissioned by accident insurance companies. The compensation for pain and suffering was between 1500 and 2000 euros per lost tooth. CONCLUSION: Traumas of incisor teeth are often followed by litigations. Clinicians performing first treatment should ensure that documentation of clinical and radiological findings is detailed and complete. This documentation plays a decisive role in medical expert opinions.

[Comparison of the osteogenic activity of bone morphogenetic protein (BMP) mutants]. / Vergleich der osteogenen Potenz gentechnisch modifizierter BMP.

BACKGROUND: Modification of the heparin binding site by alteration of the amino acid sequence of bone morphogenetic protein-2 (BMP-2) results in a change in the local retention time. The purpose of this study was to compare the osteogenic activity of T3 and T4, two mutants with increased binding capacity to heparin, and B2GDF-5 a mutant resulting from the fusion of the n-terminal amino acid sequence of BMP-2 and the c-terminal sequence of GDF-5 with wild-type BMP-2 in vivo. MATERIAL AND METHODS: The proteins were coupled to an equine-derived collagen carrier and implanted in standardized critical size calvarial defects in adult rats. After 28 days, bone formation was evaluated radiographically and the new bone was characterized histologically. RESULTS: Proteins T3 and T4 showed a higher osteogenic activity than BMP-2. Less new bone formation was observed with GDF-5 and B2GDF-5 than with-type BMP-2. No difference in bone formation was observed between GDF-5 and B2GDF-5. CONCLUSION: Increased heparin binding capacity enhances osteogenic activity of BMP-2 in vivo. This might be due to a longer retention period in the tissue and thus better bioavailability. Replacement of the N-terminal amino acid sequence of GDF-5 by the corresponding sequence of BMP-2 did not result in an increased osteogenic activity as heparin binding capacity is not the main reason for the bioavailability of GDF-5.

[Objective assessment of cosmetic outcome of scars]. / Möglichkeiten zur objektiven Beurteilung von Narben.

AIM OF THE STUDY: Cosmetic outcome of surgical treatment in the facial region is influenced by the appearance of the scar. The purpose of this investigation was to evaluate the objectivity and validity of various methods for the assessment of scars. MATERIAL AND METHODS: 45 patients undergoing facial surgery were examined three months after surgery. Three maxillofacial surgeons evaluated the wounds in a blinded manner using a visual analog scale (VAS). The patients also used the VAS for evaluation of their own scar. The surface of the scars was also measured by optical profilometry. Finally, the colour of the scars was analysed using both the RGB colour model and the HSV colour model. RESULTS: Interestingly, there was no significant correlation between the values of R (red) as assessed by the three surgeons. However, there was a very strong correlation between the values of H (hue) as assessed by the three raters. The appearance of the surface was more important in the patients' evaluation than in the raters' assessment. CONCLUSION: The hue of a scar as measured in terms of the H value in the HSV colour model is an objective and reliable measure for assessing the cosmetic outcome of facial surgery.

[Mandibular reconstruction with autologous bone and osseoinductive implant in the Göttingen minipig]. / Unterkieferrekonstruktion mit autologem Knochen und einem induktiven Implantat beim Göttinger Minischwein.

BACKGROUND: Direct mandibular reconstruction with an autologous bone transplant was compared with an osteoinductive implant following an extensive continuity resection of the lower jaw in Göttinger mini-pigs. METHOD: In nine full-grown mini-pigs a one-sided continuity defect (5 cm) was created in the lower jaw. In four animals it was filled with a 50 x 25 x 15 mm(3) collagenous carrier enhanced by rhBMP-2 (400 micro g/cm(3) rhBMP-2). In two animals only the carrier was implanted as a control. Three animals received the resected autologous bone as a free transplant. Bone regeneration and consolidation of the defects was analyzed radiographically and histologically. RESULTS: Following implantation of the osteoinductive implant, complete osseous consolidation of the continuity defect in the lower jaw was observed in all animals. The defects were completely filled with a biomechanically stable bone which showed signs of functional adaptation. The replantation of the orthotopic autologous bone did not lead to functional stability quickly enough. In the periphery only an incomplete bony bridge was formed which was interrupted by large pseudarthrosis. No consolidation of the defects was found in the control group (carrier alone). CONCLUSION: Direct reconstruction of an extensive, biomechanically loaded defect with an osteoinductive implant proved to be the superior method. The osseous regeneration observed shows an immediate functional orientation. The necessity for extensive adaptive remodeling is thus minimized.

[Evaluation of the osteoinductive potential of genetically modified BMP-2 variants]. / Evaluation der osteoinduktiven Potenz von gentechnisch modifizierten BMP-2-Varianten.

BACKGROUND: The alteration of the N-terminal amino acid sequence of BMP-2 allows modification of heparin binding of the new protein. This leads to a change in the local retention time at the site of implantation. Mutants with increased (T3, T4) and with no binding (EHBMP-2) to heparin were assessed for their osteoinductivity in vivo and compared with the wild type BMP-2. METHODS: Cylindrical collagenous carriers (diameter = 5 mm, height = 10) were loaded with different concentrations (0.25-4 micro g) of the proteins. Following intramuscular implantation into the hind legs, the bone formation was measured in radiographic follow-ups. After 28 days the newly formed bone was characterized histologically. RESULTS: Elimination of the heparin binding leads to massive reduction in osteoinductivity. On the other hand, an increase in the heparin binding leads to enhancement in the osteoinductive properties, resulting in faster bone formation with a higher yield. CONCLUSION: It could be shown for the first time that modifications of BMP-2 by gene technology can lead to proteins with enhanced binding to components of the extracellular matrix. The resulting prolonged retention time at the implantation site results in an increased osteoinductivity compared with the wild type.

Influence of platelet-rich plasma (PRP) on osteogenic differentiation of rat bone marrow stromal cells. An in vitro study.

Recent clinical reports suggest that the application of an autologous blood plasma enriched with thrombocytes by centrifugal concentration (platelet-rich plasma: PRP) can enhance the formation of new bone. There are very fewin vitro or in vivo studies published on the efficiency of PRP. In this project a three dimensional cell culture system was used to compare PRP and rhBMP-2 in vitro. Marrow derived bone forming cells from Spraque-Dawley (SD) rats were seeded on porous collagenous carriers (d=5mm, h=3mm) at a density of 4 x 10(4) cells/carrier and exposed to different concentrations of PRP (platelet counts from 2.5 x 10(8)-1.6 x 10(7) platelets/culture), rhBMP-2 (300 ng) or plasma poor in thrombocytes (platelet-poor plasma, PPP). Cultures without additional supplements were used as controls. During a culture period of 21 days cell proliferation, alkaline phosphatase activity (ALP) and calcium content (days 18, 21) were measured in 3 day intervals.PRP showed a dose dependent stimulation of cell proliferation, while reducing ALP activity and calcium deposition in the culture. BMP-2 led to an opposite cell response and induced the highest ALP activity and mineral deposition. These data suggest that PRP inhibited osteogenic differentiation of marrow derived pre-osteoblasts in a dose dependent manner. PRP is not a substitute for BMP-2 in osteogenic induction.

[Effect of periosteum on induced bone formation by autolyzed, antigen-extracted, allogeneic bone. Determination of the extent of bone formation using quantitative computerized tomography]. / Einfluss des Periosts auf die induzierte Knochenneubildung durch autolysierten, antigenextrahierten, allogenen Knochen. Bestimmung des Ausmasses der Knochenneubildung mittels quantitativer Computertomographie.

Autolyzed, antigen-extracted, allogenic (AAA) bone is an osteoinductive preparation of completely demineralized bone matrix. It has been clinically applied for years. In an experimental study in rabbits, we evaluated the influence of cortical bone and periosteum on the amount of bone formation following augmentation with AAA bone. Two implants of standardized size were placed on the femoral bone of rabbits. A cell-excluding PTFE membrane was wrapped circularly around the femur as well as the anterior implant shielding the implant from the surrounding periosteum. The posterior implant was exposed directly to the periosteum while being shielded from the cortical bone by the membrane. Thus, two compartments were created selectively, preventing contact between the periosteum or cortical bone and the implants. For each compartment the area and volume of the induced new bone were evaluated by computerized radiograph analysis and quantitative CT (pQCT) scans. Implantation of AAA bone led to new bone formation in both compartments. Contact of the periosteum and the implant led to an almost four-fold increase in bone volume. Although bone formation showed interindividual variations, the difference of both compartments was highly significant using the Student's t-test for paired samples (P < 0.0001). The data show that periosteum is the primary source of new bone formation in augmentations with osteoinductive materials as it is rich in inducible progenitor cells and is well vascularized. In osseous augmentations with AAA bone, the periosteum should be preserved in order to achieve a close contact of the osteoinductive implant. Shielding from the periosteum, e.g., by cell-excluding membranes, leads to significantly less bone formation following implantation of AAA bone and should therefore be avoided.

[Effect of different factors on the bone forming properties of recombinant BMPs]. / Einfluss unterschiedlicher Faktoren auf die knochenbildenden Eigenschaften von rekombinanten BMPs.

The extent of BMP-induced new bone formation is mainly determined by the number of mesenchymal target cells in the recipient bed as well as by the biological half-life of the BMP molecules within the tissue. While the number of inducible cells is determined by the age and vascularization of the tissue, the retention time of the BMP molecules can be influenced. One possibility is the coupling of BMPs to suitable carriers, which significantly increases the osteoinductive effect. The reason for this is the physical binding of BMPs to the carrier material, which delays the resorption of the proteins. Other factors are the composition of the carrier materials, their structural stability, and possible dislocations of carrier particles. The local tissue concentration of BMPs can also be increased by an enhanced binding of the proteins to the extracellular matrix. A BMP-2 mutant (BMP-2xa) was produced by the specific modification of the amino acid sequence using recombinant technologies. BMP-2xa induces heterotopic bone formation at significantly lower concentrations than natural BMP-2. Furthermore, BMP-2xa-induced bone tissue possesses a higher bone density.

[Comparative studies of sinus floor elevation with autologous or allogeneic bone tissue]. / Vergleichende Untersuchungen zur Sinusbodenelevation mit autogenem oder allogenem Knochengewebe.

In 63 patients, 82 elevations of the maxillary sinus were performed. As augmentation, materials autografts from the iliac crest (combined with alveolar ridge augmentations in 16 sinus lifts) were transplanted in 39 cases and osteoinductive, allogeneic bone powder (AAA bone (autolyzed, antigen-extracted, allogeneic bone): n = 8, DFDBA (demineralized freeze-dried bone allograft) and/or Grafton (demineralized bone matrix gel): n = 35) were used in 43 cases. Some 4-6 months after implantation, osteoinductive, allogeneic (demineralized) bone implants showed radio-opaque areas as an equivalent of bone formation. Histological examinations revealed that osteoinductive implants were completely transformed into patients' own bone tissue. The average augmentation height after autograft transplantations was 14 (+/- 3) mm in comparison with 9 (+/- 3) mm after allograft implantations. Histologically as well as radiologically no differences of the bone quality could be determined between the two augmentation materials. Endoscopic controls showed, in both groups, nonirritated mucous membranes. On an average 2 endosseous implants (Bone Lock or ITI-screw implants) were inserted into the augmentated maxillary sinus floors in both groups. No osseointegration was achieved in 4 out of 67 dental implants when bone autografts were used and in 2 out of 74 dental implants of the allogeneic bone group. Patients with bone autografts suffered from postoperative complaints on an average of 19 (+/- 9) days (without consideration of 2 patients with postoperative complaints persisting for more than 90 days). The average postoperative complaints of recipients of allogeneic bone implants continued for 3 (+/- 5) days. The 13 patients who underwent an ambulant sinus lift procedure with allogeneic bone powder were already symptom-free several hours after the operation. Under critical consideration of all investigated parameters, osteoinductive bone implants are preferable to iliac bone autografts for maxillary sinus augmentations in those cases in which no additional alveolar ridge augmentation is required.

[EHBMP-2. Initial BMP analog with osteoinductive properties]. / EHBMP-2. Erstes BMP-Analog mit osteoinduktiven Eigenschaften.

For the first time a non natural BMP-variant (EHBMP-2) with osteoinductive properties was produced by expression in E. coli through specific mutation of the amino acid sequence. The substitution of 12 N-terminal amino acids by a nonsense sequence results in a neglectible affinity of EHBMP-2 to the extracellular matrix. In vitro EHBMP-2 induces dose-dependent cartilage formation in neonatal muscle tissue. Single intramuscular implantation in mice results in the formation of an ossicle with functional active bone marrow. The size of the ossicle depends on the amount of implanted EHBMP-2 and can significantly be increased by the combination with a collagen carrier. The largest bone formation is observed after injection of EHBMP-2 containing collagen suspensions. In rats a stronger osteoinductive activity can be achieved by coupling of EHBMP-2 to collagen discs than by coupling natural BMP-2 to the same collagen carrier. Critical size defects in rats' mandibular angels can be restored by the combination of granular collagenous bone matrix (ICBM) with EHBMP-2. Further investigations have to show whether the altered pharmacokinetics of EHBMP-2 has advantages regarding its therapeutical use and tissue-engineering.

Inductive properties of recombinant human BMP-2 produced in a bacterial expression system.

Recombinant human BMP-2, produced in E. coli, refolded and concentrated to a purity of more than 98%, has been demonstrated to be biologically active. In vitro, amounts of 0.4 microg BMP-2 or more induced new cartilage formation in 27 out of 47 samples of a neonatal muscle tissue assay, with chondroneogenesis occurring 14 days after a four-hour contact between BMP-2 and the muscle tissue. In vivo, BMP-2 was implanted in the thigh muscle of ICR mice for a period of three weeks. Amounts of 4 microg BMP-2 and more showed heterotopic bone formation in 15 out of 17 samples. When BMP-2 was combined with a collagen carrier, amounts of 0.4 microg protein or more induced heterotopic bone formation in 30 out of 33 samples four weeks after the implantation in the abdominal wall of Sprague-Dawley rats. The results show that the E. coli-derived BMP-2 was active in different assay systems in concentrations equal to those required with mammalian cell-expressed BMP-2. It could also be demonstrated that a single morphogen (BMP-2) is enough to initiate the differentiation process associated with bone induction. The presented bacterial expression system also offers the opportunity to produce large quantities of recombinant BMP-2 for clinical applications.

[Clinical application of osteoinductive implants in craniofacial surgery]. / Klinischer Einsatz osteoinduktiver Implantate in der kraniofazialen Chirurgie.

Autolyzed, antigen-extracted, allogeneic bone (AAA bone) is prepared from cortical bones of human organ donors. AAA bone possesses osteoinductive properties as it delivers BMPs from its bone matrix. Within a prospective study, 37 cranial defects were reconstructed using AAA bone implants over a period of more than 7 years. The patients were followed-up at standardized intervals. Roentgenographic assessments and bone scintigraphies revealed osseous integration and remodelling of the AAA bone implants. In one quarter of the cases re-entry was performed 10 to 18 months after the cranioplasty (removal of osteosynthesis material, recurrence of tumor). All nine AAA bone reconstructions showed bleeding surfaces and bony integrations. A bone biopsy was taken from the center of one of these AAA bone implants and this showed new bone formation originating from the surface of the implant. In one case an AAA bone implant was lost due to infection. This is noteworthy as in approximately one third of the cases the bone implants were in direct contact with the frontal sinus. The clinical results clearly emphasize the therapeutical benefit of AAA bone for cranioplasties. Large AAA bone chips from human skull bones facilitate the reconstruction of the skull's convexity, especially when sterolithography-based operation planning is performed.

[Biological activity of E. coli expressed BMP-4]. / Biologische Aktivität von E.-coli-exprimiertem BMP-4.

BMP-4 is physiologically present in low concentrations in human bone matrix. So far the protein has only been produced in small quantities by expression in mammalian cell cultures. In this study we investigated the biological activity of E. coli-expressed BMP-4. In vitro neonatal rat muscle tissue was incubated together with BMP-4 during 4 h, followed by an incubation period of 14 days on cellulose acetate membranes in BMP-free medium. The addition of 0.4 microgram BMP-4 induced cartilage formation in 1/8 samples while 4 micrograms BMP-4 showed chondroneogenesis in 2/10 samples. When the BMP-4 concentration was increased to 40 micrograms, new cartilage formation was seen in 5/7 samples. In vivo BMP-4 was implanted intramuscularly for 3 weeks in ICR mice. Amounts of 10 micrograms rhBMP-4 and more (up to 100 micrograms) constantly induced heterotopic ossicle formation. BMP-4 was also combined with a collagen carrier and implanted for 2 and 4 weeks in the abdominal muscle of SD rats. While 0.4 microgram BMP-4 showed no bone or cartilage formation, the amount of 40 micrograms BMP-4 showed new heterotopic cartilage formation, followed by endochondral ossification in almost all samples. The results prove that E. coli-expressed BMP-4 possesses the same inductive properties as mammalian-cell-expressed BMP-4.