Abstract
The continuous presence of recombinant human bone morphogenetic protein 2 (rhBMP-2) inside a scaffold may be crucial to the outcome in bone tissue engineering. This study investigated whether the release of the growth factor rhBMP-2 via different continuous application schemes influences histomorphological aspects of the hard and soft tissues induced. Three-dimensionally printed hydroxyapatite scaffolds were implanted into one latissimus dorsi muscle of 42 female Lewis rats. Simultaneously implanted mini-osmotic pumps were used to provide a continuous application of rhBMP-2 over 1, 2, or 4 weeks (total dose 200 μg). A reference group received rhBMP-2 at the time of implantation only, and a control group received only block implantation. Bone density and histological examinations were performed after 8 weeks. No significant difference in bone density was found between the groups; however, the blood vessel count differed significantly between the groups receiving continuous treatments and both the control group and simultaneous rhBMP-2 treatment group ( P < 0.0001). Soft tissue types were distributed differently among the study groups. RhBMP-2 application via mini-osmotic pumps is as suitable for inducing bone formation as a single application at the time of implantation. The time interval over which rhBMP-2 was administered had no impact on the amount of new bone formation, probably due to the study duration and low local concentrations of growth factor.
The functional and aesthetic rehabilitation of patients suffering from critical-size bone defects involving the facial skeleton remains a great challenge in the field of reconstructive surgery. Autologous bone grafts still represent the gold standard and widely preferred therapy, but the use of such grafts is associated with an increased operation time and additional donor site morbidity. In this context, tissue-engineered bone grafts represent a promising approach to this issue.
Among other techniques in bone tissue engineering, endocultivation aims to grow individually shaped vascularized bone grafts within customized scaffolds at extraskeletal sites. Due to the presence of its own arteriovenous vessel system, the latissimus dorsi muscle is the preferred site to grow bone replacements and can ultimately provide a free tissue flap transfer with an embedded engineered bone graft.
Although techniques in bone tissue engineering are constantly being improved, most studies have reported excessive bone growth outside the scaffold margins. Furthermore histological examinations have concluded that the characteristics of the newly formed bone are comparable to those of cancellous bone, leading to less stability of these bone grafts. Excessive heterotopic bone formation might theoretically be avoided by improving the biocompatibility of the scaffold materials, thereby directly influencing the function and behaviour of adhering osteogenic cells. Besides the scaffold material, the additional application of growth factors (e.g. bone morphogenetic proteins (BMPs)) plays a crucial role in bone induction processes.
In previous trials of endocultivation, BMPs have been applied simultaneously with scaffold implantation. Knowledge about the optimal time point at which to load the scaffold with growth factors is limited and is based mainly on empirical studies. Bone morphogenetic protein 2 (BMP-2) has been delivered via pre-loading of the scaffold simultaneously with scaffold implantation, or by delayed injection into the scaffold to allow for prior soft tissue ingrowth.
In addition to pharmacologically based drug delivery systems, an implantable pump represents another option for administering BMPs at a continuous flow rate. Mini-osmotic pumps are available and have been used widely in animal studies for the administration of various types of drug. These miniature infusion pumps (3 × 0.7 cm in size) are designed specifically for small animal studies. Miniature pumps allow for controlled and continuous drug delivery at the site of interest over a specified time period.
This study evaluated the use of mini-osmotic pumps for the continuous application of recombinant human BMP-2 (rhBMP-2) in endocultivation. It was hypothesized that the continuous application of rhBMP-2, in comparison to the simultaneous injection of rhBMP-2 at scaffold implantation, might have a different impact on hard tissue formation and, furthermore, might have measurable effects on the tissue morphology inside the scaffolds.
Materials and methods
Animals and surgical procedure
Ethical approval was obtained for this animal study. Forty-two female Lewis rats (Charles River Laboratories International, Wilmington, MA, USA), at an average age of 3 months and with an average weight of 220–240 g, were used in this study. Food and water were available ad libitum. The animals were kept in a room with a controlled environment and a circadian day–night rhythm of 12 h. All interventions were performed under general anaesthesia, through intraperitoneal injection of 0.2 μl/g body weight xylazine (Rompun, 2%; Bayer Healthcare, Leverkusen, Germany) and 1 μl/g body weight ketamine (Ketavet, 10%; Pfizer Pharma, Berlin, Germany).
After local disinfection of the shaved skin (Kodan; Schülke & Mayr, Norderstedt, Germany), an incision was made with a scalpel over the thoracic spine ( Fig. 1 A ), following which a subcutaneous blunt dissection was performed. A pouch was prepared in the latissimus dorsi muscle in which to implant a three-dimensionally printed scaffold ( Fig. 1 B–D).
Twenty-four animals (eight animals per group) were chosen for the implantation of mini-osmotic pumps (Alzet; Durect Corporation, Cupertino, CA, USA). After scaffold implantation, another pouch was prepared on the left side of the animal’s back. A tube leading into the centre of the scaffold was connected to the mini-osmotic pump ( Fig. 1 E, F), which was then placed into the separate pouch. The mini-pumps were set to release their contents over the course of 1, 2, or 4 weeks after activation (for a total dose of 200 μg). The wound was closed with resorbable sutures (Vicryl; Ethicon, Norderstedt, Germany). Another group of animals (reference group, n = 9) did not undergo mini-pump implantation, but received 200 μg rhBMP-2 simultaneously with scaffold implantation. A control group of animals ( n = 9) underwent scaffold implantation, but did not undergo mini-pump implantation and did not receive any rhBMP-2. The group allocation and study protocol is further summarized in Fig. 2 .
Weekly intraperitoneal fluorochrome injections were started at 2 weeks after implantation and were continued until the end of the trial. The fluorochromes applied were Xylenol Orange (6% in NaHCO 3 , 5 ml/kg; Sigma-Aldrich, Steinheim, Germany), Alizarin (3% in 2% NaHCO 3 , 0.8 ml/kg body weight; Sigma-Aldrich), doxycycline (1 mg/kg; Ratiopharm SF, Ulm, Germany) and Calcein Green (1% in 2% NaHCO 3 , ; Sigma-Aldrich). The specimens were assessed for the presence of each fluorochrome in all specimens, in a semi-quantitative manner: 0, not detected; 1, some detected; 2, widely detected, as described previously.
Conventional computed tomography examinations
Conventional computed tomography (CT) examinations were performed to evaluate bone density (Somatom; Siemens AG, Munich, Germany; settings of 120 kV, 210 mAs, and 46.82 mGy). A radiologist who was unfamiliar with the group allocation performed all examinations. The bone density value per animal was determined in the area of scaffold implantation. Scaffold areas were marked manually on each slice for every animal using the CT scanning software provided by the manufacturer. The bone density was measured in Hounsfield units (HU).
Sample preparation and histology
Animals were sacrificed after an 8-week observation period by CO 2 insufflation. The scaffold-bearing muscular pouch was then excised. The specimens were cut in half before embedding in wax or methyl methacrylate (MMA). Additional histopathological techniques for sample preservation and staining were routinely applied, as described previously.
In brief, after decalcification and dehydration in a graded series of alcohol solutions, the first half of each specimen was embedded in wax and then sliced to a width of approximately 5 μm. Histological staining was then performed (Masson–Goldner and haematoxylin–eosin). The other half of each specimen was embedded in MMA and sliced to a width of 100 μm for microradiography, 40 μm for fluorescence microscopy, and 10 μm for toluidine staining, respectively. Microphotographs were then obtained (Mikrophot-FXA; Nikon, Düsseldorf, Germany). Photoshop CS5.1 (Adobe Systems, Munich, Germany) was used for further image processing.
Histological assessment
To standardize the histological evaluations, the cross-sectional area of the scaffold was divided into four major zones ( Fig. 3 ).
