Clin Oral Invest DOI 10.1007/s00784-017-2050-1

ORIGINAL ARTICLE

Application of tissue-engineered bone grafts for alveolar cleft osteoplasty in a rodent model Paula Korn 1 & Maria Hauptstock 1 & Ursula Range 2 & Christiane Kunert-Keil 3 & Winnie Pradel 1 & Günter Lauer 1 & Matthias C. Schulz 1

Received: 14 July 2016 / Accepted: 4 January 2017 # Springer-Verlag Berlin Heidelberg 2017

Abstract Objectives The clinical standard for alveolar cleft osteoplasty is augmentation with autologous bone being available in limited amounts and might be associated with donor site morbidity. The aim of the present study was the creation of tissueengineered bone grafts and their in vivo evaluation regarding their potential to promote osteogenesis in an alveolar cleft model. Materials and methods Artificial bone defects with a diameter of 3.3 mm were created surgically in the palate of 84 adult Lewis rats. Four experimental groups (n = 21) were examined: bovine hydroxyl apatite/collagen (bHA) without cells, bHA with undifferentiated mesenchymal stromal cells (MSC), bHA with osteogenically differentiated MSC. In a control group, the defect remained empty. After 6, 9 and 12 weeks, the remaining defect volume was assessed by cone beam computed tomography. Histologically, the remaining defect width and percentage of bone formation was quantified. Results After 12 weeks, the remaining defect width was 60.1% for bHA, 74.7% for bHA with undifferentiated MSC and 81.8% for bHA with osteogenically differentiated MSC. For the control group, the remaining defect width measured * Paula Korn [email protected]

1

Department of Oral and Maxillofacial Surgery, Faculty of Medicine BCarl Gustav Carus^, Technische Universität Dresden, Fetscherstr. 74, 01307 Dresden, Germany

2

Institute for Medical Informatics and Biometry, Faculty of Medicine BCarl Gustav Carus^, Technische Universität Dresden, Fetscherstr. 74, 01307 Dresden, Germany

3

Department of Orthodontics, Faculty of Medicine BCarl Gustav Carus^, Technische Universität Dresden, Fetscherstr. 74, 01307 Dresden, Germany

46.2% which was a statistically significant difference (p < 0.001). Conclusions The study design was suitable to evaluate tissueengineered bone grafts prior to a clinical application. In this experimental set-up with the described maxillary defect, no promoting influence on bone formation of bone grafts containing bHA could be confirmed. Clinical relevance The creation of a sufficient tissueengineered bone graft for alveolar cleft osteoplasty could preserve patients from donor site morbidity. Keywords Alveolar cleft . Bovine bone substitute . Histomorphometry . Mesenchymal stromal cells . Tissue-engineered bone grafts

Introduction One of the most common hereditary craniofacial anomalies in humans is cleft lip and alveolar cleft with or without cleft palate. In literature, the prevalence is stated with approximately 1 in 700 live births with variability depending on geographic origin, ethnic groups and environmental as well as socioeconomic factors [1]. An essential part of the surgical treatment is the secondary alveolar cleft osteoplasty before the eruption of the permanent canine teeth at a patient’s age of nine to eleven years. The augmentation of the persisting alveolar bone defect closes the oronasal fistula, provides osseous support for the dentition and stabilizes the dental arch as well as the local soft tissue [2]. If the alveolar cleft osteoplasty is not performed and the alveolar bone defect remains untreated, a delayed or misguided tooth eruption might occur. This can lead to functional and aesthetic limitations for the patient. Currently, autologous bone grafts, e.g. from the iliac crest, are the clinical standard for alveolar cleft osteoplasty [3].

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The transplantation of autologous bone leads to another surgically created wound in the donor site area which might be associated with intra- and postoperative complications. Furthermore, the availability of autologous bone is still limited [4–6]. This led to an intensive search for alternative materials to replace or supplement the autologous bone graft for alveolar cleft osteoplasty. In oral surgery, bone augmentation in non-critical size defects is clinically established. Especially after cyst enuclation, tooth extraction and before insertion of dental implants, bone augmentation is applied routinely [7, 8]. There are different groups of bone substitute materials which are all clinically approved. According to their origin, they are classified into autologous, allogenic, xenogenic and alloplastic materials. However, the exclusive application of bone substitution materials in critical size defects is clinically not recommendable [9]. For larger defects, the challenge is that the use of pure substitute materials is not sufficient to induce a stable defect bridging by formation of new bone. One reason is the poor cellular ingrowth into substitute materials replacing extensive bony defects. Furthermore, possible interferences of dental eruption and/or jaw development limit the application of pure materials. The method of tissue engineering could be an option for the preparation of bone grafts with the ability to promote the osseous healing of large defects [10]. Currently, in bone tissue engineering, the main cell source are bone marrow-derived mesenchymal stem cells [11, 12]. They have a high osteogenic potential and were characterized in numerous studies. Nevertheless, an in vitro cell expansions is always necessary prior to in vivo application [12]. Besides the chosen cells, the scaffold material is important for the creation of a tissueengineered bone graft. The scaffold has to enable cell adhesion and proliferation before and after transplantation. Furthermore, it should be biocompatible, osteoconductive, mechanically stable and resorbable in an adequate time [13]. Gladysz et al. reviewed the current literature regarding stem cell-based regenerative therapy for alveolar cleft reconstruction. Besides the required characteristics, there is no consensus which scaffold materials is the best suitable for this application. Ceramics, like calcium phosphate and HA, synthetic and natural polymers are discussed and each group has specific advantages to improve the scaffold-cell interaction, which seems to be a precondition for better clinical results [14]. Against the background that sintered bovine hydroxyl apatite (bHA) is a clinically widely used bone substitution material, it is of clinical interest whether the material is also suitable for the creation of tissue-engineered bone grafts to promote the ossification in maxillary defects. In vitro studies analysing bHA with osteoblast-like cells indicated a high in vitro biocompatibility and a sufficient expression of characteristic proteins and transcription factors [15, 16]. The in vivo biocompatibility of bHA is well documented for

non-critical size defects, e.g. sinus floor augmentation [17]. There are only few studies analysing pure bHA in critical size defects wherefore the suitability could not yet be confirmed clearly [18]. Currently, to the best of our knowledge, no experimental study has evaluated tissue-engineered bone grafts containing bHA in large defects like an artificial alveolar cleft. The hypotheses of the present experimental study are as follows: (1) It is possible to create bone grafts of bHA scaffolds and mesenchymal stromal cells in vitro. (2) The surgically created bone defect of 3.3 mm diameter in the maxilla of adult Lewis rats will not reunite within the experimental period of 12 weeks. (3) The in vivo application of the different tissue-engineered bone grafts containing undifferentiated or osteogenically differentiated MSC leads to a significant improvement of defect ossification within the healing time of a maximum of 12 weeks. The defect ossification was assessed by radiological and histological analysis ex vivo.

Methods and materials Animal model The study design was approved by the Commission for Animal Studies at the District Government Dresden, Germany (AZ: 24(D)-9168.11-1/2013-7). Adult male Lewis rats (Janvier Labs, Le Genest-Saint-Isle, France) with an average body weight of 460 g and an age of six to 8 months at the beginning were chosen for this experimental study. The animals (n = 84) were housed in a light- and temperaturecontrolled environment. They had access to food and water ad libitum. Scaffolds For this tissue engineering application bovine, deproteinized hydroxyl apatite combined with 10% collagen (bHA, BioOss® Collagen, Geistlich Biomaterials, Wolhusen, Switzerland) was chosen. The commercially available 100 mg biomaterial blocks (5 mm × 5 mm × 10 mm; size of the single hydroxyl apatite particle 0.25–1 mm) were cut into slices with a diameter of 3.3 mm and height of about 1 mm under sterile conditions. Due to the collagen content, the scaffold material could be deformed in a moist environment and adapted to the bony defect easily. Cell culture and in vitro analysis In general anaesthesia, initiated by intraperitoneal injection of 100 mg/kg body weight ketamine (Riemser Arzneimittel AG, Greifswald, Germany) and 10 mg/kg xylazine (PharmaPartner Vertriebs-GmbH, Hamburg, Germany), the femur of donor rats (adult, male Lewis rats) was removed and

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subsequently, the animals were killed in a carbon dioxide bath. For isolation of the mesenchymal stromal cells (MSC), the femur was separated in the area of the epiphysis and the bone marrow was aspirated under sterile conditions. Centrifugation of the aspirate at 1200 rounds per minute (rpm) for 10 min as well as aspiration of the supernatant and resuspension with minimum essential medium (MEMα (Gibco®, Thermo Fisher Scientific Inc., Waltham, USA)) followed. Afterwards, the cells were transferred into culture flasks (T-75 cell culture flask, Greiner Bio-One, Frickenhausen, Germany) and placed into an incubator (Hera Cell, Heraeus Kulzer GmbH, Hanau, Germany) at 37 °C and 5% CO2. The haematopoietic stromal cells which were marked by a CD34 antibody (antibodies-online, Aachen, Germany) could be removed from the eluate by negative selection using the MiniMACS™ Separators (Miltenyi Biotec GmbH, Bergisch Gladbach, Germany). The remaining MSC were cultivated in vitro with changing of the culture medium every 3 days for a period of 7 to 14 days until a confluence of 95% was achieved. Cells of the second passage were used for further tissue engineering application. For colonization, the portioned biomaterial was placed into a 24-well plate and incubated with 2 ml culture medium for 24 h at 37 °C. In preparation of the cell colonization, the scaffolds were washed with phosphate-buffered saline without Ca 2+ and Mg 2+ (PBS, Life Technologies Cooperation, Darmstadt, Germany). MSC of the second passage were washed with 2 × 6 ml PBS too and then detached using 2 ml of trypsin/EDTA. The mobilized cells were taken up in specific medium (undifferentiated MSC: MEMα; osteogenically differentiated MSC: Opti-MEM® (Thermo Fisher Scientific Inc.)) and passed over into a 1.5-ml falcon tube. After centrifugation at 8000 rpm for 5 min at room temperature, the removal of the supernatant and resuspension with 1 ml medium was performed. The measurement of the cell number was carried out using a Casy® Cell Counter and an associated analysis system (Roche Diagnostics GmbH, Mannheim, Germany). Then, the substitute material was colonized with cell-medium-suspension containing about 200,000 cells each. Additional cell culture medium was added after 2 h so that the initial adherence of the cells to the bone substitute material was ensured. Depending on the experimental group, different mediums were used on the colonized scaffolds for the final cultivation for 3 days before in vivo insertion. In one group, undifferentiated MSC were planned. Thus, the colonized scaffolds were cultivated applying MEMα. Another experimental group should contain osteogenically differentiated MSC. The differentiation of the cells into osteoblasts was performed using the medium Opti-MEM® supplemented with 10% foetal calf serum (Gibco®, Thermo Fisher Scientific Inc.), 50 μM ascorbic acid (Sigma-Aldrich, St. Louis, USA), 10 mM βglycerophosphate (Sigma-Aldrich) and 10 μM dexamethasone (Sigma-Aldrich) [19]. In both groups, the tissue-

engineered bone grafts were stored in an incubator at 37 °C with 5% CO2 and 95% humidity for 3 days. The successful differentiation into osteoblasts was confirmed using an Alkaline Phosphatase Kit (Sigma-Aldrich, Taufkirchen, Germany) according to the manufacturer’s instructions. Qualitative results were obtained by light microscopy. For further characterization of both cell lines, MSC as well as osteogenically differentiated MSC, the gene expression for bone morphogenetic protein 4 (BMP-4) and the stem cell factor runt-related transcription factor 2 (Runx2) were quantified after 3, 7 and 12 days cultivation by quantitative polymerase chain reaction (PCR). The isolation of the total RNA from cells was done using the standard protocol of the RNeasy Mini Kit (Qiagen, Hilden, Germany). After determination of the RNA concentration, an amount of 200 ng total RNA was transcribed reversely using innuScript Reverse Transcriptase, inNucleotide Mix and Random primer (Analytik Jena AG, Jena, Germany). To quantify the expression of different genes, gene-specific TaqMan PCR primers and probes (Bmp4: Rn00432087_m1; Runx2: Rn01512296_m1; Rat ACTB (actin, beta) endogenous control: 4352931E) were obtained from PE Applied Biosystems (Weiterstadt, Germany) and quantitative real-time PCRs were performed using a TOptical cycler (Analytik Jena AG) as well as the innuMix qPCR MasterMix Probe (Analytik Jena AG). Absolute copy numbers of the studied genes and 18S cDNA were determined using calibration curves generated with cloned PCR fragment standards. Copy numbers of individual transcripts are given in relation to those of 18S cDNA. Each series of experiments was performed twice and Bno-template controls^ with water were carried out parallely in all experiments as described previously [20]. The analysis of the tissue-engineered bone grafts was completed by a scanning electron microscopy examination (XL30 ESEM; Environmental Scanning Electron Microscope, Koningklijke Philips N.V., Eindhoven, Netherlands). The cellular colonization of the scaffolds was visualized with a magnification up to 500 under humid conditions.

In vivo application of the bone grafts The rats were anaesthetized by intraperitoneal injection of ketamine (100 mg/kg body weight) and xylazine (10 mg/kg body weight) and fixed in a dorsal position. A simulated alveolar cleft was created surgically in the anterior maxilla of each animal. First, a sagittal incision was made following the mid-palatal suture. After elevation of a mucosal flap and removal of the periosteum, a localized bone defect with 3.3 mm in diameter was created using a diamond-coated cylindrical shaped drill (DiT Dental-Instrumente GmbH, Oberlungwitz, Germany). According to the randomized distribution, each rat received one bone graft (Fig. 1):

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Fig. 1 Overview of the study design. Each seven animals were sacrificed after 6, 9 and 12 weeks according to the schedule. Alizarin was applied 7 days and calcein 3 days prior to sacrifice

Group I: Control, no bone graft (n = 21) Group II: bHA without cells (n = 21) Group III: bHA with undifferentiated MSC (n = 21) Group IV: bHA with osteogenically differentiated MSC (n = 21)

After insertion of the bone graft, the flap was repositioned and wound closure was performed using 5-0 Ethilon suture (Ethicon, Norderstedt, Germany). Postoperatively, the animals received amoxicillin trihydrate (Fort Dodge Veterinär GmbH, Würselen, Germany) 15 mg/kg body weight once and 4 mg/kg body weight carprofen (Sigma-Aldrich) every 24 h for 4 days. All drugs were injected subcutaneously. The animals were fed a soft diet for the first 3 days and, subsequently, received a regular diet. Postoperatively, the animals and their behaviour were monitored and the body weight was measured every 2 weeks. For the ex vivo assessment of the dynamic bone formation, all rats received intraperitoneal injections of the fluorochrome dyes alizarine (20 mg/kg body weight) and calcein (30 mg/kg body weight) 7 and 3 days prior to sacrifice. Sample preparation The animals (seven per group) were sacrificed after 6, 9 and 12 weeks of healing time. After cone beam computed tomography, the cranium was dissected and fixed in 4% formaldehyde for 48 h. As described previously after dehydration in a graded series of ethanol, all samples were embedded in methylmethacrylate (Technovit® 9100, Heraeus Kulzer, Wehrheim, Germany) [21]. Coronal sections were produced according to Donath’s sawing and grinding technique [22]. Thus, the 4 central sections of each specimen could be achieved for evaluation. Subsequently, the sections measuring 60 μm in thickness were polished. After analysis of the fluorochrome marker uptake, Masson-Goldner trichrome staining followed. Histological analysis All samples were imaged by fluorescence microscopy and, after staining, by light microscopy (Olympus BX 61, Olympus Deutschland GmbH, Hamburg, Germany) and cell ^ F Imaging Software for Life Science (Olympus).

Multiple image alignment was performed using an automatic scanning table (Märzhäuser, Wetzlar, Germany). Thus, eight images per sample were scanned with a 10 × 10-fold magnification and manually fused to one image. Fluorochrome marker uptake was analysed to assess the dynamics of bone formation at the defect margins. The histological analysis focused on the structure of bone, the soft tissue and the resorption of bone grafts in the defect area. For histomorphometrical evaluation, the remaining width of the defect was measured between the cranial and caudal defect margins being located in the cortical bone. The remaining defect width was related to the individual initial defect width which could be clearly identified by differences in bone morphology from the newly formed bone. Additionally, the content of newly formed bone in the whole defect area was measured and is displayed as a percentage value. Formulas for histomorphometrical measurement: Remaining defect width [%] = ((cranial distance of newly formed defect margins [mm] + caudal distance of newly formed defect margins [mm]) ÷ 2) ÷ (cranial distance of initial defect margins [mm] + caudal distance of initial defect margins [mm]) ÷ 2)* 100 Bone formation [%] = (area of newly formed bone [μm2] ÷ area of the defect [μm2]) * 100

All measurements were realized by one examiner who was masked regarding to the experimental group. Formulas for histomorphometrical measurement are as follows: Cone beam CT Immediately after sacrifice, cone beam computed tomography (3D Acciutomo, J. Morita MFG. Corp., Kyoto, Japan) of the cranium was performed applying a tube voltage of 70 kV and an amperage of 4 mA as described previously [23]. All data was transferred to the workstation of a commercially available navigation system (BrainLAB AG, Feldkirchen, Germany) as standard digital imaging and communications in medicine (DICOM) files. The artificial alveolar cleft was visualized on the workstation monitor in multiplanar image reformations for each animal using the image-data processing software of the navigation system. The remaining defects were outlined on 16

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slices per defect area (slice interval 0.25 mm) using drawing tools. A three-dimensional reconstruction of the defect was performed and the volumes of the remaining cleft defects were calculated using a navigation software (iPlan 2.6 Cranial, BrainLAB AG). To eliminate inter-operator variability and to avoid mistakes in outlining the region of interest, reformatting and measurements were performed by a single examiner who measured all samples twice. Statistical analysis Statistical analysis was performed using SPSS Statistics 23.0.0.0 (IBM Cooperation, Armonk, USA). A mean value was calculated for each defect resulting from the different sections. For all experimental groups, mean and standard deviations were calculated. For data in all experimental groups and for all healing times, a normal distribution could be assumed. The influence of the fixed factors Bexperimental group^ and Bhealing time^ was tested with an ANOVA with additional consideration of the influences of the various individuals as a random factor. Bonferroni-adjusted multiple comparisons were done as post hoc tests.

Results In vitro results The removal and cultivation of the MSC was realizable without limitations. The osteogenic differentiation of the MSC could be demonstrated by alkaline phosphatase analysis (Fig. 2a). Scanning electron microscopy visualized the surface structure of the bone grafts where the attached cells could be clearly identified (Fig. 2b). Both groups of cells were analysed regarding the mRNA content for BMP-4 after 3, 7 and 12 days of cultivation. Osteogenically differentiated MSC showed the highest mRNA amount of BMP-4 after 7 days of cultivation before a significant reduction to 79.9% (at experimental day 12) occurred. Additionally, the runt-related transcription factor 2 (Runx-2) was quantitatively determined. The mRNA expression of Runx2 was 2.2-fold and 1.7-fold increased in osteogenically differentiated MSC compared to undifferentiated MSC. During the cultivation, the Runx2 mRNA decreases in differentiated cells continuously, whereas undifferentiated cells do not show any differences in the mRNA expression of both, BMP-4 and Runx2. Results are depicted in Fig. 3. Clinical results Eighty of 84 animals completed the study. This represents a survival rate of 95.2%. Three animals of the following groups died during surgery: bHA + osteogenically differentiated

Fig. 2 Alkaline phosphatase analysis of osteogenically differentiated cells after 3 days of cultivation, magnification 100× (a). Scanning electron microscopy of bovine hydroxyl apatite/collagen colonized with undifferentiated mesenchymal stromal cells after 3 days of cultivation on the scaffold, magnification 500× (b)

MSC (6 weeks healing time) and bHA + undifferentiated MSC (9 and 12 weeks healing time). One animal died within the healing time (bHA + osteogenically differentiated MSC; 12 weeks healing time). It was not necessary to kill animals before the end of the study due to their health condition. During the healing time, all animals showed no disturbance regarding their behaviour and wound healing. The food and water intake was not compromised. After an initial loss of body weight postoperatively, the weight remained stable.

Radiological results A cone beam CT of each defect area was made ex vivo and analysed regarding the remaining defect volume. All experimental groups showed a reduction of the defect volume after 12 weeks healing time compared to the first measurement after 6 weeks (Table 1). This was found to be statistically significant for the control group from 6 to 12 weeks (p = 0.005) and bHA + osteogenically differentiated MSC

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from 6 to 9 weeks (p = 0.022). The comparison between the experimental groups showed a significant smaller defect for the control group than the bHA + ostegeonically differentiated MSC after 12 weeks (p = 0.001). The application of pure bHA led to a statistically significant smaller defect volume compared to bHA + osteogenically differentiated MSC after 6 weeks (p = 0.044).

Descriptive histology

Fig. 3 Quantification of bone morphogenetic protein 4 (BMP-4) (a) and runt-related transcription factor 2 (Runx2) (b) mRNA levels in undifferentiated (grey columns) and differentiated rat mesenchymal stromal cells (checked columns) by real-time PCR at different time points. The mRNA levels of all tested genes are given in relation to that of actin, beta. Means ± standard deviation are given in all cases for n = 8–9 samples. Stars indicating significant differences between differentiated and undifferentiated cells at a fixed time: *p < 0.05, **p < 0.01, ***p < 0.005, and significant differences between differentiated cells at different times: # p < 0.05, ##p < 0.01, ###p < 0.005; unpaired t test Table 1 Results of the cone beam computed tomography: remaining defect volume (mm3)

Experimental group

control

bHA

bHA + undiff. MSC

bHA + osteo. diff. MSC

Polychrome fluorescence labelling The application of the fluorescence labels alizarin and calcein were performed 7 and 3 days prior to sacrifice of the animals. Alizarin was only detectable in some ex vivo specimens whereas calcein led to a sufficient labelling of the mineralized tissue being formed at the time of application. The control group without bone graft exposed a symmetrical osteogenesis at both defect margins beginning at the former surgically created defect margins and continuing into the direction of the defect centre. The distance between alizarin and calcein labels was homogenous which can be assumed as a consistent bone formation (Fig. 4a). At the caudal area of the defect, appositional bone formation occurred unrelated to the artificial defect due to a periosteal reaction. After 9 weeks, the intensity of bone formation decreased, especially at the caudal defect margins. The osteogenesis was focused on a coneshaped region in direction of the defect centre. With continuing healing time, the defect margins became rounded and the fluorescence labels occurred with less extent and intensity. Sometimes, discontinuations of the labels were detectable due to remodelling processes at the defect margins.

Healing time (weeks)

6 9 12 6 9 12 6 9 12 6 9 12

Number

7 7 7 7 7 7 7 6 6 6 7 6

Remaining defect volume (mm3) Mean

SD

13.43a 12.57 11.07a,c 12.43d 14.07 12.57 14.07 13.75 13.25 14.50b,d 12.43b 14.08c

0.89 1.21 2.32 1.90 0.93 1.17 1.40 1.04 1.48 2.17 0.89 1.36

The analysis is based on the number of 80 animals and the values are displayed as mean and their standard deviation Between italic values marked with the same letter, a statistically significant difference was measurable (a p = 0.005, b p = 0.022, c p = 0.001, d p = 0.044)

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Fig. 4 Polychrome fluorescence labelling (magnification 10 × 10, multiple image alignment). Alizarin (red) was applied 7 days and calcein (green) 3 days prior to sacrifice. The control group showed homogenous osteogenesis on both defect margins after 6 weeks of healing time (a). After 9 weeks osseous integration of bovine hydroxyl apatite granulate could be observed in a particular case (b)

The experimental group with pure bHA bone grafts showed mainly the same characteristics as the control group regarding the localization and quantity of the fluorescence labels. Osteogenesis started at the surgically created sharp defect margins and went into the direction of the defect centre, likewise. The bHA granules did not expose mineralization processes on their surface which could be detected with the chosen magnification. After 9 weeks, an osseous integration of the granules close to the defect margins occurred sporadically (Fig. 4b). The labels were generally reduced compared to earlier time points after 12 weeks, indicating decreasing mineralization intensity with proceeding osseous healing. Thinned fluorescence labels and smaller cone-shaped osteogenesis areas were the result of the application of bone grafts containing MSC. Initial resorption occurred on the granules after 12 weeks. Between undifferentiated and osteogenically differentiated MSC, no differences regarding dynamics and quantity of bone formation could be found by this evaluation method. Histomorphology Four samples of each animal displaying the bone defect were included into the analysis. The main result of this analysis was that no complete bony reunion of the artificial defect occurred in any experimental group. According to the details of the local bone formation, differences depending on the applied bone grafts were obvious. The control group showed a symmetrical formation of woven bone in cone-shaped bone spiculae with a thin layer of osteoid on both defect margins, whereas the defect itself was filled with fibrous tissue. The fibrous tissue exposed a more and more organized structure

with ongoing healing time. With proceeding time, the bone spiculae showed a centripetal growth. However, no bridging of the defect occurred. On the basis of the cone adult lamellar bone dominated. Woven bone and osteoid were located in the middle and peak area of the cone. After 12 weeks, no osteoid was detectable anymore and the cone became more rounded (Fig. 5a). On the caudal defect margin periosteal bone formation occurred. The application of pure bHA led to a similar cone-like formation of bone spiculae with an increasing broad basis of lamellar bone and woven bone in the peak area. In contrast to the control group, this experimental group showed an earlier maturation of the newly formed bone. The bHA granules were embedded in fibrous tissue and mainly located in the centre of the defect. In one particular case, the biomaterial was integrated into the bone. In general, the osteogenesis was oriented cranially in the direction of the nasal septum (Fig. 5b). The tissue-engineered bone grafts did not enhance the osteogenesis, irrespectively of which cells were used for the colonization of the material (Fig. 5c, d). In comparison to the control group, the size of the cone-like bone areas seemed to be reduced at all three time points. After 12 weeks, the content of osteoid was increased for bone grafts containing undifferentiated MSC compared to pure bHA or scaffolds colonized with osteogenically differentiated MSC indicating a proceeding bone formation. In both groups, osteogenesis occurred from the defect margin in the direction to the middle of the defect, likewise. After 6 weeks, in areas of direct contact between host bone and bone graft resorptions with formation of Howship’s lacunae were visible. These were limited to the host bone whereas the biomaterial showed no signs of resorption. During the healing time, the bHA granules were increasingly integrated into fibrous tissue without any alteration of their morphology. In some cases, several granules were embedded in one capsule of multilayered fibrous tissue. All experimental groups had in common that in particular cases a destruction of the caudal part of the nose septum occurred due to surgical preparation of the defect in the upper jaw. However, no influence on the local bone formation in the defect area was detectable. Furthermore, sometimes bHA granules dislocated into the surrounding tissue. If this happened in an area of mechanical stress, e.g. onset of muscles, local resorption on the particular bone resulted. A dislocation into the nasal cavity did not cause histomophologically detectable alterations. Histomophometry: remaining defect width The control group exposed a remaining defect width of 63% related to the individual initial defect width after 6 weeks with a statistically significant reduction after 12 weeks (46% p = 0.001) (Table 2). After 12 weeks, all experimental groups containing bone grafts showed a statistically significant larger

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Fig. 5 Descriptive histology after 12 weeks of healing. Exemplary images of all experimental groups: control group (a), pure bovine hydroxyl apatite (b), bone grafts containing undifferentiated mesenchymal stromal cells (c), bone grafts containing osteogenically differentiated mesenchymal stromal cells (d). No complete osseous

healing of the defect occurred, but starting from the defect margin, cone-like bone formation is detectable in all experimental groups. In b– d, the formerly compact scaffold is reduced to single hydroxyl apatite granulae, which are not osseointegrated. (Masson-Goldner trichrome staining, magnification 10 × 10, multiple image alignment)

defect compared to the control (Table 3). At the end of the experimental study, the remaining defect width measured 60% for pure bHA, 75% bHA + undifferentiated MSC and 82% for bHA + osteogenically differentiated MSC. Apart from the bHA + osteogenically differentiated MSC, the other groups exhibited a reduction of the defect width within the study period. For the control group, this was statistically significant (p = 0.001). The biomaterial bHA did not lead to a faster reduction of the defect width, irrespective of MSC colonization, in comparison to the control group. This was mostly statistically significant (Table 3). The application of undifferentiated MSC was associated with a

smaller defect than the use of osteogenically differentiated MSC at the end of the study but this difference was not statistically significant. After 6 and 9 weeks, the group with osteogenically differentiated MSC exposed a smaller defect than the group with undifferentiated MSC which was statistically significant (p = 0.044) after 9 weeks.

Table 2 Results of histomorphometric analysis: remaining defect width related to the individual defect size after surgery (%) and percentage of newly formed bone in relation to the defect size

Experimental group

control

bHA

bHA + undiff. MSC

bHA + osteo. diff. MSC

Histomophometry: percentage bone formation After 6 weeks, the control group exposed 25% newly formed bone in the defect area (Table 2). With proceeding healing

Healing time (weeks)

6 9 12 6 9 12 6 9 12 6 9 12

Number

Remaining defect width (%)

Bone formation (%)

Mean

SD

Mean

SD

7

62.6a

7 7 7 7 7 7 6 6 6 7 6

k

17.4 19.1 16.7 19.7 18.4 14.5 16.4 10.5 12.9 17.3 11.9 9.7

24.5c,d 32.9c,e 43.0d,e 22.8f,g 30.2f 30.8g 8.9h 10.1i 20.5h,i 17.8 22.0j 11.5j

12.9 14.6 13.3 12.8 14.0 12.1 7.3 7.8 10.9 12.5 11.8 7.3

58.9 46.2a,k 63.3 63.6 60.1 81.5 82.7 74.7 73.6 71.8b 81.8b

The analysis is based on the number of 80 animals and the values are displayed as means and their standard deviation Between italic values marked with the same letter a statistical significant difference was measurable within one experimental group (a p = 0.001, b p = 0.047, c p = 0.018, d p < 0.001, e p = 0.003, f p = 0.038, g p = 0.018, h p < 0.001, i p = 0.002, j p = 0.002, k p = 0.006). For statistical inter-group analysis, see Tables 3 and 4

Clin Oral Invest Table 3 Pair wise comparisons between the values for remaining defect width in relation to the individual defect size after surgery (%) with regard to the different healing times. Between italic values a statistically significant difference (p<0.05) was measurable

Experimental groups

Healing time 6 weeks Sign.

Control

bHA

bHA + undiff. MSC

bHA + osteo. diff. MSC

bHA

1.000

1.000

0.004

0.000

0.000

0.000

bHA + osteo. diff. MSC Control

0.048 1.000

0.008 1.000

0.000 0.004

bHA + undiff. MSC bHA + osteo. diff. MSC

0.000 0.060

0.000 0.257

0.002 0.000

Control bHA

0.000 0.000

0.000 0.000

0.000 0.002

bHA + osteo. diff. MSC Control

0.241 0.048

0.044 0.008

0.568 0.000

bHA

0.060

0.257

0.000

bHA + undiff. MSC

0.241

0.044

0.568

Discussion Children suffering from a congenital cleft alveolus need local bone augmentation in the area of the cleft to stabilize the dental arch and to enable the eruption of the permanent canine [2]. Currently, autologous bone from the iliac crest is considered as golden standard for the augmentation. Due to reduced availability and to possible donor site morbidity, there are efforts to implement the current developments of bone tissue engineering to create a sufficient alternative to autologous bone grafts [14]. The present study analysed the application of in vitro manufactured bone grafts containing bHA and MSC in a model of alveolar cleft with regard to their potential to promote local bone formation.

Experimental groups

Control

bHA

bHA + undiff. MSC

bHA + osteo. diff. MSC

12 weeks Sign.

bHA + undiff. MSC

time, the value increased statistically to 33% (p = 0.018) and 43% (p < 0.001). The application of pure bHA led to comparable values after 6 (23%) and 9 weeks (30%), also with statistically significant increase (p = 0.038). However, after this time, the osteogenesis seemed to reach a steady state and no further bone formation occurred. In combination with MSC, no additional effect of the local bone formation was measurable. In contrast, both groups with cells had a lower percentage of bone formation, which was statistically significant at certain points (Table 4). The lowest rate of bone formation with 11% was shown by the group of bHA + osteogenically differentiated MSC after 12 weeks which represents a statistically significant reduction of bone formation from week 9 to the end of the study (p = 0.002).

Table 4 Pair wise comparison between the values for percentage bone formation in relation to the defect size (%) with regard to the different healing times. Between italic values a statistically significant difference (p<0.05) was measurable

9 weeks Sign.

Healing time

bHA bHA + undiff. MSC bHA + osteo. diff. MSC Control bHA + undiff. MSC bHA + osteo. diff. MSC Control bHA bHA + osteo. diff. MSC Control bHA bHA + undiff. MSC

6 weeks Sign.

9 weeks Sign.

12 weeks Sign.

1.000 0.000 0.161 1.000 0.000 0.528 0.000 0.000 0.010 0.161 0.528 0.010

1.000 0.000 0.001 1.000 0.000 0.034 0.000 0.000 0.001 0.001 0.034 0.001

0.000 0.000 0.000 0.000 0.004 0.000 0.000 0.004 0.023 0.000 0.000 0.023

Clin Oral Invest

Animal model Currently, the complexity of a congenital human alveolar cleft or palate cannot be displayed by any pre-clinical model. Therefore, experimental studies are limited to artificial bone defects in different models. The established small animal model is the rat, because in its maxillary bone a cleft-like defect can be prepared surgically [24, 25]. An alternative large animal model would be the maxillary bone of dogs [26]. Genetically, it is also possible to create cleft models [27]. Due to a gene deletion, an incomplete development of the palate and alveolus as well as other pathologies occurred in a mice model. The gene defect led to perinatal lethality. This model is similar to the sevoflurane-induced embryonic cleft model, not suitable to evaluate new bone grafts [28]. In the present study, the adult male Lewis rat was chosen due to the possibility to transplant cells interindividually without immunological disadvantages. Both analysing methods confirmed that an initial bone defect of 3.3 mm led to a defect without osseous bridging within the 12 weeks of healing observed in the present study. In vitro behaviour of the bHA bone graft Tissue engineering is one approach to create bone grafts in vitro and apply them in vivo to promote bone regeneration. The basis is the assortment of suitable scaffold materials and cells which were in accordance with the intended clinical result. The in vitro findings of the present study confirmed the possibility to use bHA in the context of tissue engineering applications [29, 30]. The biocompatibility and cytotoxity of bHA were analysed by Liu et al. using human osteoblasts, likewise [31]. The colonized biomaterial underwent cell vitality staining and four different tests of biocompatibility. Thus, the suitability of the material could be confirmed. Clinically, these findings were completed by numerous studies evaluating bHA for various clinical applications [32–34]. The bone marrow of adult mammals represents a sufficient source for multipotent stem cells [35]. These cells have the potential to differentiate into bone cells, cartilage cells, muscle cells, ligament cells, tendon cells, adipose cells and stroma cells. In the context of the study with the aim to promote bone formation in a maxillary defect, osteogenic differentiation of the MSC was chosen. The osteogenic differentiation could be realized sufficiently and the following RNA analysis showed a higher level of gene expression for BMP-4 in osteogenically differentiated MSC compared to undifferentiated cells. Physiologically, BMP-4 promotes the differentiation of MSC as osteoprogenitor cells into osteoblasts [36]. The highest level of BMP-4 expression occurred in vitro after 7 days of cultivation. Cells, like MSC, can be verified by stem cell markers like Runx2. Runx2, an essential transcription factor in the development of the skeletal system [37], is required for

osteoblast cell transition from proliferative to differentiated cells [38–40]. Runx2 expression is followed by the upregulation of genes expressed in osteoblasts like osteocalcin, Alpl and collagen type I [41]. Runx2-deficient mice are devoid of osteoblasts and, consequently, of bone tissue [42]. Mastrangelo et al. compared the expression of alkaline phosphatase, osteocalcin and Runx2 of osteogenically differentiated MSC cultivated on bHA or synthetic hydroxyl apatite [15]. The measured level of protein expression was higher on bHA indicating its suitability for further tissue engineering applications. In vivo behaviour of the bHA bone graft In conclusion of the measurements using cone beam CT, the control defect exposed a statistically significantly smaller defect volume than tissue-engineered bone grafts containing osteogenically differentiated MSC. Between pure bHA, grafts with undifferentiated MSC and the control group no statistically significantly differences occurred, likewise. The defect volume decreased with proceeding healing time but statistically significantly only for the control group. Histomorphometrically, the remaining defect width was evaluated. Again, the control defect showed the lowest values compared to the other experimental groups for this parameter. After 12 weeks, the control defect was statistically significantly reduced compared to all other groups. It has to be highlighted that control and pure bHA groups showed similar rates of bone formation and defect width after 6 and 9 weeks. However, during further healing time, a stagnation of bone formation was detectable for pure bHA. Meanwhile, the bone formation in the control group proceeded to grow statistically significantly. Moreover, bone grafts containing undifferentiated MSC showed a continuing statistically significant osteogenesis after 12 weeks. In contrast, the application of osteogenically differentiated MSC led to a significantly reduced bone formation rate with ongoing healing time. In the context of the initial hypothesis of the study, it has to be concluded that the application of tissue-engineered bone grafts containing bHA and MSC did not enhance the bone formation in this maxillary defect in a rodent model. The quantity of bone formation and thus, the reduction of the defect size decreased in the given order at the end of the study period: control group > pure bHA > bHA + undifferentiated MSC > bHA + osteogenically differentiated MSC. An unexpected finding of the study is that no enhancement of defect ossification could be shown in the artificial defect. One possible reason might be the application of a non-resorbable bone substitute. After degradation of the collagen scaffolds, the remaining particles might have been exposed to micromovements, thus hampering a sufficient ossification of the defect. Furthermore, the defect was located directly posterior of the maxillary incisors in an area where chewing forces might occur.

Clin Oral Invest

Postoperatively, the animals were provided with soft diet to avoid chewing pressure on the wound area. However, a total wound rest might not have been achieved causing micromovements, likewise, which is a drawback of the study. In a clinical situation, nasal gastric tubes might have been applied to avoid stress during the wound healing period. However, this is not possible in an animal model. The application of a thin biocompatible membrane to cover the biomaterial might avoid this in further animal studies [43]. Comparison of bHA bone grafts The current state of research regarding tissue-engineered bone grafts is extensive but just a few studies analysed these bone grafts in alveolar cleft osteoplasty models. Mayer et al. evaluated a copolymer scaffold (poly(lactide-co-glycolide)) with and without recombinant BMP-2 in a maxillary defect in a dog model [44]. Also, collagen and hydroxyl apatite scaffolds were combined with recombinant BMP and analysed regarding their influence on local bone formation in a rat model [43, 45]. The mentioned studies had in common that the bone grafts led to a positive effect on bone formation. Nevertheless, the scaffolds were not colonized with cells. Thus, a direct comparison with the present study is not reasonable. Pourebrahim et al. applied hydroxyl apatite/ tricalciumphosphate scaffolds which were in vitro colonized with osteogenically differentiated MSC in a cleft defect in dogs [26]. The tissue-engineered bone grafts were compared with autologous bone grafts. The mean percentage of bone regeneration was measured after 15 and 60 days. Local bone formation was significantly higher for autologous bone grafts. Nevertheless, the analysed combination of hydroxyl apatite/ tricalciumphosphate and osteogenically differentiated MSC was recommended as an alternative material for grafting of this maxillary defect. The mentioned studies abstained from testing the pure scaffold material or empty control defect like other studies did. For example, BONITmatrix® is a clinically established bone substitute material consisting of synthetic hydroxyl apatite and tricalciumphosphate. In 2014, a study analysing the effect of the colonization with either undifferentiated or osteogenically differentiated MSC on local bone formation in a maxillary defect of the rat was published. The study included a control group as well as one group with the pure scaffold for comparison [21]. The undifferentiated MSC showed a higher potential to reduce the defect size by ossification compared to the other bone grafts. In comparison with the present study, undifferentiated MSC seemed to be more efficient than osteogenically differentiated MSC, likewise. Both studies led to oppositional results regarding the control defect. In the cited study, the remaining defect volume and width were the highest for the control group. In contrast, the present study led to statistically significant reduced values for this experimental group. Reasons for the contrary results

might be seen in the applied biomaterial (synthetic hydroxyl apatite/tricalciumphosphate vs. bHA) and the chosen healing times (up to 6 weeks vs. up to 12 weeks). Both biomaterials differed regarding numerous material associated parameters. Thus, differences in the experimental and clinical performance are expectable. Synthetic and bHA are very stable and will be resorbed slowly. It represents an initial placeholder for local osteogenesis starting from the defect margins of the host bone. Later, the material will be integrated into the newly formed bone [16]. Due to its stability hydroxyl apatite is applied frequently and with sufficient results. In particular cases, an inhibiting effect of hydroxyl apatite on initial osseous healing of extraction sockets could be found [46]. In the present study, this inhibiting influence of pure bHA was detectable after 12 weeks because up to 9 weeks, the bone formation was similar to the control and then a stagnation was measurable. To what extent this observation in a large maxillary defect is transferable to other defects, e.g. extraction sockets, is uncertain. Synthetic tricalciumphosphate is less stable than hydroxyl apatite, therefore, degraded more rapidly than Thus, it is often combined with hydroxyl apatite to improve its material characteristics. In the context of the study, no promotion of osteogenesis in a large maxillary defect was detectable and it has to be concluded that the combination of synthetic hydroxyl apatite/tricalciumphosphate might be more efficient than the application of bHA independently of the application of cells. Translation into clinical application Due to the promising results for the implementation of tissueengineered bone grafts as an alternative to autologous bone which were gained in several small and large animal studies, first studies in humans were realized. Van Hout et al. reviewed controlled, randomized clinical trials analysing tissueengineered bone grafts [47]. Just three studies fulfilled the mentioned criteria until 2011 [48–50]. Different resorbable scaffolds made of collagen where combined with BMP-2 and led to comparable values for volume and newly formed bone. The authors recommended further studies to analyse the quality of the newly formed bone in the defect area. The clinical application of bHA for alveolar cleft osteoplasty was evaluated in a study with 23 patients by Benlidayi et al. [51]. There was no statistically significant difference between defects grafted with bHA or autologous bone. The clinical application of pure bone substitute materials for large defects, e.g. congenital clefts, was discussed critically because the eruption of the permanent canine can be disturbed [10]. Due to their regenerative potential, MSC are one option for tissue engineering applications. Related to alveolar cleft osteoplasty, the suitability of these cells was analysed by Pradel et al. and Behnia et al. [23, 52]. The combination of the biphasic scaffold of hydroxyl apatite/tricalciumphosphate, osteogenically

Clin Oral Invest

differentiated MSC and platelet-derived growth factor led to a defect ossification of 51% after 6 months [23]. Nevertheless, the study abstained from comparing the results with the clinical standard, the autologous bone graft [52]. Pradel et al. measured a defect ossification of 41% for bone grafts of demineralized bone matrix and osteogenically differentiated MSC after 6 months. The transplantation of autologous spongious bone from the iliac crest resulted in 37% ossification of the cleft [23]. The currently published studies regarding the application of tissue-engineered bone grafts for alveolar cleft osteoplasty have in common that the number of cases is limited and all of them recommended further studies to identify potential bone grafts. A future trend in bone tissue engineering for cleft alveolar osteoplasty might be the implementation of 3D printing of individual scaffolds based on preexisting computed tomography scans of the particular patient. Berger et al. demonstrated a successful preparation of individual tricalciumphosphate-polyhydroxybutyrate scaffolds colonized with human MSC in vitro [53]. As a next step, these kinds of bone grafts have to be analysed in vivo to evaluate their potential to promote osteogenesis in maxillary defects.

Acknowledgements The authors want to thank Mrs. Diana Jünger for her extensive assistance in preparing the bone grafts. Furthermore, the authors are grateful to Dr. Roland Jung and the team of the Experimental Centre of the Medical Faculty BCarl Gustav Carus^, Technische Universität Dresden, for the care of the animals and assistance during the surgical interventions. Dr. Heike Meißner is acknowledged for the scanning electron microscope images. Moreover, the authors thank Mr. Torsten Jannasch for the assistance in preparing the images. Compliance with ethical standards Conflict of interest The authors declare that they have no conflict of interest. Funding The study was financially supported by an internal research grant BMeDDrive Projekt Start^ of the Faculty of Medicine BCarl Gustav Carus^, Technische Universität Dresden, Germany. The bone substitute was kindly provided by Geistlich Biomaterials, Wolhusen, Switzerland. Ethical approval This article does not contain any studies with human participants performed by any of the authors. All applicable international, national and/or institutional guidelines for the care and use of animals were followed. Informed consent required.

For this type of study, formal consent is not

Conclusion For patients suffering from congenital alveolar cleft, the development of bone grafts as an alternative to autologous bone from the iliac crest would be advantageous. The method of tissue engineering enables the creation of in vitro bone grafts consisting of clinically established biomaterials and living cells. Before these bone grafts can be analysed in humans, pre-clinical studies are necessary to identify potential combinations of biomaterials and cells leading to a high degree of defect ossification. The present study examined tissueengineered bone grafts of bHA and MSC with regard to their effect on the promotion of local osteogenesis in an animal model of alveolar cleft. The following conclusions can be drawn from the study: (I) The in vitro preparation of bone grafts containing bHA and MSC, both undifferentiated and osteogenically differentiated, was possible. (II) An artificial bone defect of 3.3 mm represented a non-reuniting defect in the maxilla of adult Lewis rats within the entire experimental period of 12 weeks. (III) The study could not confirm that bone grafts of bHA enhance ossification in a model of alveolar cleft. This was independent of the application of MSC. (IV) Undifferentiated MSC were more efficient compared to osteogenically differentiated MSC regarding the enhancement of defect ossification after 12 weeks of healing. (V) Future studies should focus on resorbable scaffolds in the maxillary defect model to allow osteoconduction in the defects. Also, the implementation of new technologies like 3D printing of defect-specific scaffolds should be considered to improve the initial interaction between tissue and biomaterial.

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