Cell based bone tissue engineering in jaw defects

Biomaterials 29 (2008) 3053–3061

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Biomaterials journal homepage: www.elsevier.com/locate/biomaterials

Leading Opinion

Cell based bone tissue engineering in jaw defectsq Gert J. Meijer a, *, Joost D. de Bruijn b, c, Ron Koole a, Clemens A. van Blitterswijk c a

Department of Oral Maxillofacial Surgery, University Medical Centre Utrecht, Heidelberglaan 100, 3584 CX Utrecht, The Netherlands Department of Materials, Queen Mary University of London, Mile End Road, London E1 4NS, United Kingdom c Institute for Biomedical Technology, University of Twente, Building Langezijds (LA), P.O. Box 217, 7500 AE Enschede, The Netherlands b

a r t i c l e i n f o

a b s t r a c t

Article history: Received 20 February 2008 Accepted 15 March 2008 Available online 22 April 2008

In 6 patients the potency of bone tissue engineering to reconstruct jaw defects was tested. After a bone marrow aspirate was taken, stem cells were cultured, expanded and grown for 7 days on a bone substitute in an osteogenic culture medium to allow formation of a layer of extracellular bone matrix. At the end of the procedure, this viable bone substitute was not only re-implanted in the patient, but also simultaneously subcutaneously implanted in mice to prove its osteogenic potency. In all patients, a viable bone substitute was successfully constructed, which was proven by bone formation after subcutaneous implantation in mice (ectopic bone formation). However, the same construct was reluctant to form bone in patients with intra-oral osseous defects (orthotopic bone formation). Although biopsies, taken 4 months after reconstructing the intra-oral bone defect, showed bone formation in 3 patients, only in 1 patient bone formation was induced by the tissue-engineered construct. Although bone tissue engineering has proven its value in animal studies, extra effort is needed to make it a predictable method for reconstruction jaw defects in humans. To judge its benefit, it is important to differentiate between bone formation induced by cells from the border of the osseous defect (osteoconduction) in relation to bone matrix produced by the implanted cells (osteogenesis). Ó 2008 Elsevier Ltd. All rights reserved.

Keywords: Bone tissue engineering Cell culture Osteogenesis Stem cell

1. Introduction Since Friedenstein’s first publications in the 1980s [1], it was realized that mesenchymal stem cells (MSCs) can be utilized to engineer mesenchymal tissues, such as bone and cartilage. Therefore, scientists worldwide are working to provide the right carrier and the appropriate set of cells that, once re-transplanted, will ensure bone repair. Caplan et al. combined mesenchymal stem cells with a scaffold to allow paracrine and host derived factors to produce bone matrix after implantation. The proof of concept was convincingly shown in ectopic rodent models [2–12] as well as in critical size defects in rodents [10,13–16]. Also in larger animals orthotopic application proved to be feasible in multiple models, such as in segmental femur defects in dogs [17] or sheep [18,19]. In addition, bone formation in reconstructed skull [20] and mandibular defects [21] in sheep, and iliac wing defects in goats [22] were observed. In

q Editor’s Note: Leading Opinions: This paper is one of a newly instituted series of scientific articles that provide evidence-based scientific opinions on topical and important issues in biomaterials science. They have some features of an invited editorial but are based on scientific facts, and some features of a review paper, without attempting to be comprehensive. These papers have been commissioned by the Editor-in-Chief and reviewed for factual, scientific content by referees. * Corresponding author. E-mail address: [email protected] (G.J. Meijer). 0142-9612/$ – see front matter Ó 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.biomaterials.2008.03.012

contrast to more than 300 animal studies, only a few case reports of successful reconstructions in humans have been published [23–31]. The present study was conducted to prove that, in concordance to the successful outcome of earlier animal studies in our group [16,22,32–34], autologous mesenchymal stem cells are also capable of bone formation in humans. Intra-oral defects, which needed to be reconstructed to allow dental implant placement, are ideal test sites, because, before preparing the implant bed to install dental implants, a biopsy of the reconstructed area can be easily taken, implicating no extra burden for the patient [35,36]. Moreover, all surgical procedures can be performed under local anaesthesia. No effort was made to select patients with a specific type of intra-oral defect. The only criterion was that reconstruction of these bone defects was indicated in preparation for dental implant placement in a secondary stage. In order to test if the cultured samples indeed have the capacity of bone formation, in a synchronously conducted control study, each patient cultured samples were subcutaneously placed in the back of mice with non-cultured samples as control.

2. Methods 2.1. Patients Initially, 10 patients, who were scheduled for reconstruction of various intra-oral osseous defects, were included in this study. Repair of these defects was indicated as

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preparation for dental implant placement in a secondary stage. All patients gave written consent. This study was conducted in accordance with the Medical Ethics Committee from the University Medical Centre of Utrecht, The Netherlands. 2.2. Creation of the viable bone substitute 2.2.1. Harvest and transport of human bone marrow aspirates To collect the bone marrow in each patient the cortical bone at the right posterior iliac crest was perforated using a trocar. From this opening 3  3–5 ml of bone marrow aspirate was taken, while the biopsy needle was placed under different angles for every aspirate. In total, 10–15 ml of bone marrow was collected. This procedure was performed in sterile manner under local anaesthesia. After harvesting, the bone marrow cells were cultured within 24 h of harvesting and transported at room temperature in heparinised tubes to avoid clotting. 2.2.2. Expansion and determination of osteogenic potential of human mesenchymal stem cells (HMSCs) Following transportation, the cells were counted and seeded in tissue culture plates at a concentration of 100.000 nuclear cells/cm2. The HMSCs were cultured as described in the literature [3,6,14,22,37]. The culture medium contained a-MEM supplemented with 10% Foetal Bovine Serum (FBS), dexamethasone and antibiotics (penicillin/streptomycin), and was refreshed regularly. At near confluency, the adherent HMSCs were enzymatically released by trypsinisation in order to plate them again to obtain larger cell numbers. For all patients ‘‘replating’’ was maximally exercised 3 times. In advance of the implantation procedure, the osteogenic lineage of the cultured cells was checked by alkaline phosphatase staining. Alkaline phosphatase (ALP) is expressed by precursor-osteoblasts, which will differentiate in bone forming cells during culture time. This is the cell population of interest most likely generating the bone forming potential at the end of the culturing time. 2.2.3. Seeding of HMSCs on scaffolds; control of vitality and distribution After reaching the required amount of cells, depending on the size of the defect, the HMSCs were seeded on 1–4 mm3 hydroxyapatite particles with interconnected pores and 65% porosity (Pro-Osteon 500; Interpore, Melle, Belgium). This was done in a ‘‘static’’ way in 25 wells replica-plates with 3 particles in each well. In order to differentiate the cells towards osteoblasts the HMSCs were grown for another 7 days in a concentration of 4  108 cells/cc scaffold in fully supplemented medium in order to allow osteogenic differentiation and extracellular matrix formation. During this period the seeded HMSCs were triggered to differentiate into osteogenic cells by dexamethasone. To control the distribution and vitality of the cells before implantation, some particles were stained with, respectively, methylene blue and trypan blue. Before transportation the particles were soaked in transport medium consisting of a-MEM devoid of FBS. Finally, the viable bone substitute was packaged, transported to the operating room, rinsed with physiological salt solution (0.9% NaCl) and implanted. 2.2.4. Implantation of a viable bone substitute in mice In total, for each patient, 12 particles (6 particles in 2 mice each) were subcutaneously implanted in athymic mice. In each mouse, also 2 control particles without cells were implanted. Thus implicating that for each patient 2 mice served as control, identifying the bone forming capacity of the implanted constructs. The particles were implanted para-spinally in the back of each mouse with a minimal distance between particles of at least 3 mm. In total, 20 mice were used. All samples were explanted after 6 weeks. Subsequently histological sections were made after alcohol dehydration and methyl methacrylate embedding. 2.3. Surgical procedure 2.3.1. Application of a viable bone substitute in humans Surgery was performed under local anaesthesia. In case of augmentation, an incision was made at the bone crest and a full thickness mucoperiosteal flap was raised. The viable bone substitute was applied to the proper position and after dissecting the periosteal sheath at the base the mucoperiosteal flap was repositioned and sutured. Although the application of a semi-permeable membrane may protect the graft and reduce fibrous tissue ingrowth, no membranes were used, because of their inhibiting effect on ingrowth of vessels from the periosteum. In 2 patients a sinus floor elevation procedure was performed. According to the method described by Boyne and James [38] in the lateral sinus wall a trapezium shaped door was prepared using a diamond burr and ample cooling. Subsequently, the top hinge door was fractured inwards with a caution to preserve the sinus membrane from tearing. Hereafter, the bone substitute was inserted in the created space. 2.3.2. Insertion of dental implants Four months after application of the viable bone substitute, the operation site was opened first to take a biopsy and second to insert the dental implant. Preoperative antibiotics AugmentinÒ (GlaxoSmithKline, Pittsburgh) were administered one day before surgery. It must be emphasised that the biopsy was taken exactly on the same location, as where subsequently the dental implant was installed, thus in

the middle of the reconstructed area. After taking the biopsy with a 2 mm diameter hollow trephine drill (Straumann, Waldenburg, Switzerland), the created diameter was widened to 4 mm to allow dental implant installation, using a low-speed drill and continuous physiological salt irrigation. Implants applied were Frialit Synchro implants (Friadent GmbH, Mannheim, Germany) or 3i implants (3i Implant Innovations, Inc, Palm Beach Gardens, Florida). 2.3.3. Placement of healing caps According to the golden standard, 3 months after insertion of the dental implant in the lower jaw and 6 months in the upper jaw, a third operation was performed: the cover screws were removed and healing caps were inserted, allowing the dentist to construct a prosthetic superstructure (crown or bridge). 2.4. Histological analysis The samples were dehydrated through a graded series of ethanol under vacuum and routinely embedded in methyl methacrylate (MMA). Light microscopy sections were processed on a histological diamond saw (Leica Sf1600) and were stained with basic fuchsin/methylene blue, in order to study tissue ingrowth into the bone substitute. 2.5. Postoperative follow-up After reconstruction of the osseous defects, patients were evaluated using radiographic and clinical evaluation of functionality at 3, 6, 9, 12 and 15 months after application of the viable bone substitute. After placement of the healing caps, subsequently each 3 months, clinical assessments were performed. To evaluate the healthiness of the peri-implant tissues, the gingival index [39], the supra-gingival plaque index [40] and a dichotomous bleeding index were used. An OrthoPantomoGram or intra-oral X-ray was taken according to standard procedures at each follow-up interval and at 15 months after surgery. The height of the reconstructed area was measured and corrected for distortion due to magnification. Changes due to osteolysis and peri-implant bone loss were scored. 2.6. Role of the funding source The sponsors of the study had no role in study design, data collection, data analysis, data interpretation, or writing of the report. The corresponding author had full access to all the data in the study and had final responsibility for the decision to submit for publication.

3. Results 3.1. Patients Initially, 10 patients, who were scheduled for reconstruction of intra-oral osseous defects, were involved in this study, 4 male and 6 female. Age of patients varied from 18 to 48 years with an average of 28.5 year. The first patient was enrolled in November 2000. With a follow-up period of 15 months, the last patient was completed in January 2003. 3.2. Creation of the viable bone substitute 3.2.1. Harvest, transport and intake of the bone marrow aspirate Aspiration was performed under local anaesthesia. No serious adverse events were observed. Except for the first, for each patient sufficient amount of cells, more than 50  106 nucleated cells/ml bone marrow were collected from the bone marrow aspirates. 3.2.2. Expanding and determination of osteogenic potential of HMSCs During the proliferation phase the HMSCs were monitored for growth rate during every trypsinisation step in the process. During the proliferation phase, patient 1 did show an insufficient growth rate, which is explained by a very poor cell concentration of the aspirate (Fig. 1). This patient refused a second aspiration procedure and therefore was excluded from the clinical study. Simultaneously to the seeding and differentiation of cells on the scaffold particles, also cells were cultured in tissue culture plates for the same time period in differentiation medium with and without dexamethasone. After 6–7 days of culture these cells were stained for alkaline phosphatase. Differentiation of the cells resulted in an

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shown in Table 2, in total, 6 defects were reconstructed with a defect size varying between 0.5 and 2.5 cc (Fig. 3). 3.3.2. Installment of dental implants In 6 patients a total of 11 dental implants were inserted. In patients 6, 7, and 8 one implant, in patient 5 two implants and in patients 9 and 10 three implants were installed (Table 2, Fig. 3). In patient 9 one implant became mobile and was subsequently removed. Subsequently, instead of solitary crowns on 3 implants, a bridge was made on 2 implants (Fig. 3). 3.4. Histological analysis

Fig. 1. Growth rates of the HBMC cultures during the proliferation phase of the manufacturing process.

obvious difference of alkaline phosphatase expression between the 2 culture conditions. It was demonstrated that more than 90% of the cultured HMSCs from all patients, except for patient 1, reacted on adding dexamethasone by showing strong positive alkaline phosphatase staining after different time points and different passages of culture. This strong ALP-reaction indicates that the cultured cells have an osteogenic capacity ‘in vitro’ (Table 1). 3.2.3. Seeding of HMSCs on scaffolds; control of vitality and distribution The methylene blue staining showed the initial distribution of cells during seeding. For all samples, the trypan blue stain confirmed that also at the moment of implantation the cells were uniformly divided over the particles and still vital (Fig. 2). 3.2.4. Implantation of viable substitute in mice; ‘‘in vivo’’ results after 6 weeks After subcutaneous implantation in athymic mice, histological evaluation of the tissue-engineered constructs related to the patients 5–10 showed all new bone formation (Table 1). In none of the control samples, without cells, any bone formation was observed. 3.3. Surgical procedure 3.3.1. Implantation of ‘‘viable bone substitute’’ in humans During dental implant placement in patient 2, it was observed that 2 out of 4 implant sites were inflamed. As a result, the protocol was adapted and starting with patient 5, prophylactic antibiotics were administered, not only before dental implant placement, but also in advance of applying the viable bone substitute. In order to achieve an uniform protocol for all patients in our study, the results of the clinical study will be focussed on these 6 patients (5–10). As

Table 1 ‘In vivo’ bone formation in mice after 6 weeks of subcutaneous implantation in relation to age, gender and alkaline phosphatase staining Patient

Age (years)

Gender

Alkaline phosphatase (ALP) staining

05 06 07 08 09 10

25 19 22 20 45 18

F M M M F F

þ þ þ þ þ þ

‘In vivo’ bone formation subcutaneously in mice þ Cells

 Cells

Yes Yes Yes Yes Yes Yes

No No No No No No

As 11 implants were installed, also 11 biopsies were collected. The most representative results are shown in Fig. 2. In 3 patients (patients 5, 7, and 9) the biopsies showed bone formation. In 2 of these cases (patients 5 and 9) bone formation on the scaffold material occurred nearby the pre-existing bone of the bone defect, indicating that bone was very likely formed on basis of osteoconduction alone. Only in the biopsy of patient 7 areas with ‘de novo’ bone formation more than 7 mm separated from the preexisting bone tissue was seen, which is considered strongly suggestive for osteogenesis; bone formation produced by the implanted cells (Fig. 2, Table 3). 3.5. Postoperative follow-up Follow-up showed no specific data. Oral hygiene was performed well, resulting in healthy peri-implant tissues. During the research period, 1 implant (Table 2; patient 9, location 16) was lost. The other implants were stable; no significant bone loss was observed. X-rays showed stable bone volume except for patient 9; the loss of the dental implant (location 16) could be predicted based on the ongoing reduction in vertical bone height (Table 4). 4. Discussion The use of autologous (host) bone grafts has been the preference for bone repair and regeneration [41–45]. The patient’s own bone lacks immunogenicity and provides bone forming cells, which are directly delivered at the implant site [46,47]. Moreover, autologous bone grafts recruit mesenchymal cells and induce them to differentiate into osteogenic cells through the exposure of osteoinductive growth factors [41,43,46,48]. In contrast to the favourable aspects of autologous bone, the search for alternatives has been motivated by the drawbacks of the harvesting procedure. The extra surgery causes morbidity at the donor site [41–43,46,47,49]. Postoperative continuous pain [43,50–52], hypersensitivity [43], pelvic instability [51–53], infection [46,50] and paresthesia [43,46] are possible complications associated with bone grafting which affect 10–30% of the patients [47,50]. Moreover, the amount of bone that can be collected is limited. A challenging, biological driven method is to mimic a bone graft by combining living osteogenic cells with biomaterial scaffolds ‘ex vivo’, to allow the development of a three-dimensional autologous bone substitute. Several investigators [4,32–34,54–56] have reported that bone marrow cells, when cultured under appropriate conditions, can maintain their viability and are able to differentiate into osteogenic cells. Several studies in bone tissue engineering involving cells in humans have been published in which the authors claim their technique to be successful [23–31]. The first clinical report (2001) described the preliminary results (6–13 months after surgery) of the treatment of 3 patients with various segmental defects (4 cm bone segment loss in the right

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Fig. 2. Overview for patients 5–10. First column; particles stained with methylene blue immediate after seeding, showing cell distribution. Second column; particles stained with trypan blue after 1 week of culturing, showing cell vitality. Third column; histology 6 weeks after subcutaneous implantation in mice, showing ‘in vivo’ bone formation (red line in contact with brown HA particle, black arrow). Fourth column; histology after 4 months of implantation in bone jaw defects of patients. Only patient 7 shows bone formation induced by the implanted cells (white arrow).

tibia, 4 cm in the right ulna, and 7 cm in the right humerus), using ‘ex vivo’ expanded HMSCs loaded on a three-dimensional scaffold of the shape and size of the missing bone fragment [23,24]. All 3 patients presented a repair of the fracture site: the implants

showed good integration of the newly formed bone and abundant callus formation. Also after a follow-up of 5 years the patients functioned well. The authors admitted that, due to the high density of the mineral and the relatively low porosity (50–60%), it was

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Table 2 Description of the defect, location of original teeth/molars that need to be replaced, amount of implanted bone substitute, surgical procedure, and number of dental implants placed and lost Patient number

Description of defect

Location of missing teeth/molar

Amount implanted (cc)

Surgical procedure

Dental implants placed

5

1 large defect in lower jaw

1.1

Augmentation

2 (FrialitÒ)

6 7 8 9

1 1 1 1

0.5 0.5 0.5 2.5

Augmentation Augmentation Augmentation Sinusfloor elevation

1 1 1 3

10

1 large defect in upper jaw

4.4 4.5 2.1 2.1 2.1 1.4 1.5 1.6 1.1 1.2 1.3

1.5

Augmentation

3 (FrialitÒ)

solitary defect in upper jaw solitary defect in upper jaw solitary defect in upper jaw large defect in upper jaw

very difficult to monitor the patient’s recovery during the postsurgery time using two-dimensional X-rays [25]. Recently, the authors reported that the CT-scans of the same 3 patients, made 6–7 years postoperatively, showed complete healing of the original gaps between scaffold and bone segments and also were suggestive for the presence of new bone formation within the scaffold pores [26]. Also in 2001 Vacanti et al. published a clinical case in which periosteal cells were applied in bone tissue engineering [27]. Explant cultures were performed using 8 pieces (1 cm2) of periosteum, which were harvested under sterile conditions from the patient’s distal part of the radius. After 9 weeks, 20  106 periosteal cells were injected in combination with a hydrogel (alginate) into a calcium phosphate scaffold (Pro-Osteon 500; Interpore, Melle, Belgium) to reconstruct the distal phalanx of a thumb. They concluded that the procedure resulted in the functional restoration of a stable and biomechanically sound thumb of normal length. A biopsy was taken 10 months post-surgery and the histology revealed that the scaffold–cell construct was vascularised and well integrated in the host tissue. However, only 5% of the construct showed newly formed lamellar bone and endochondral tissue. Schimming and Schmelzeisen [28] reconstructed in 27 patients the posterior maxilla using a matrix derived from mandibular periosteum cells on a polymer fleece (Ethisorb; Ethicon, Norderstedt, Germany). In 12 patients reconstruction was combined with implant placement (one-step method). Therefore, no biopsies were taken, only radiographic and clinical assessments were performed. The other 15 patients were treated according to a two-step method, as also conducted in our study. Due to a lack of primary fixation to allow dental implant placement, first reconstruction of the host area was performed, allowing to take a biopsy, 3 months later, during dental implant placement. In 8 of these 15 patients replacement resorption with connective tissue was found. In the other 7 patients, biopsies confirmed the presence of bone tissue. In a comparable clinical study, performing maxillary sinus augmentation with secondary implant placement, 2 different carriers were tested [29]. Cells of mandibular periosteum were cultured with autologous serum and then transferred onto a collagen matrix or seeded onto natural bone mineral. Biopsies taken after 6 months showed bone formation (38%) in all collagen carriers loaded by periosteal cells. With respect to the natural bone mineral carrier, after 8 months of implantation, more bone formation (32% versus 25%) was observed between samples with (n ¼ 5) or without (n ¼ 3) cultured periosteal cells. In another clinical study, bone regeneration after grafting enucleated mandibular cyst cavities using either autogenous osteoblasts cultured on a biomaterial or autogenous spongious iliac bone was tested [30]. In 9 patients 11 mandibular cysts were filled with tissue-engineered bone (autogenous osteoblasts

(3IÒ) (3IÒ) (FrialitÒ) (FrialitÒ)

Loss of dental implants

1

cultured on demineralised bone matrix). As controls 11 patients with 11 cyst were treated with spongious iliac bone. Radiographic analysis showed that in both groups bone regeneration took place in a similar fashion. No statistical significant differences were observed. The authors did not forget to mention that, also without filling the cysts, bone density increases in time [57]. Also the beneficial effect of HMSCs mixed with platelet-rich plasma (PRP) was analysed, as applied during a distraction osteogenesis procedure used for limb lengthening [31]. Although the results were preliminary, this method could shorten the treatment period by acceleration of bone regeneration during distraction genesis [31]. The above-mentioned studies indicate the possibilities of bone tissue engineering, but their results are debatable. To confirm the osteogenic capacity of the implanted cells, at least biopsies need to be taken. Solely, the use of radiographs cannot differentiate between bone tissue formed by the implanted cells or by the cells from the border of the osseous defect [23–26]. Subsequently, when biopsies confirm the presence of bone, an attempt should be made to identify if the bone is formed by the implanted cells. Based on this starting point, in our study the results of 2 biopsies were labelled as insufficient. Unfortunately in neither of the presented studies [23–31] this issue was addressed. This present study was conducted to prove if there is benefit of HMSCs seeded on a scaffold in restoring an osseous jaw defect. Although the choice of carrier material can be discussed, hydroxyapatite particles were chosen based on their osteoconductive properties. Moreover these materials are used many times by general practitioners and also support the induction of osteogenesis of MSCs [58]. As suggested in the literature alkaline phosphatase (ALP) staining is used to confirm the osteogenic capacity of cells ‘in vitro’ [59]. All cultured constructs stained positively for alkaline phosphatase (ALP) and also induced bone formation in mice after 6 weeks of subcutaneous implantation, implicating that the concept of culturing mesenchymal cells and differentiating them into osteoblasts actually does work in mice! However, exactly the same construct failed to form bone in the patients themselves. In 3 patients bone formation was observed. In 2 patients (numbers 5 and 9) bone growth was seen at the direct border of the original bone surface, indicative of osteoconduction. Implicating that probably the cells from the original bone surface caused the bone formation. In only 1 patient (number 7) bone formation was observed, that was suggestive to have been induced by the implanted cells. The biopsies in the other 3 patients (numbers 6, 8, and 10) showed no or hardly any bone formation. The key question is; why do HMSCs produce bone ectopical in mice, but are reluctant to do so in jaw defects in humans. To be successful in bone tissue engineering (1) sufficient numbers of cells

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Fig. 3. Overview for patients 5–10. First column; radiographs showing the alveolar defects. Second column; showing the reconstruction (arrow) by augmentation (5–8) and by sinus elevation procedure (9 and 10). Third column; radiographs showing the dental implants and the prosthetic construction (crown or bridge). Fourth column; clinical situation at the end of the rehabilitations (arrow).

with osteogenic capacity should be present, as also (2) an appropriate scaffold to seed the cells in combination of (3) factors to stimulate the cells ‘in vivo’ [3]. All these 3 prerequisites can be fulfilled by engineering. However, the fourth demand for success is dependent on patient factors, namely sufficient vascular supply [3]. Lack of sufficient vascular supply, resulting in immediate cell death after implantation, may be the cause for the disappointing

outcome of this study. Vascularisation is essential for cells in order to get oxygen and nutritional supply. Osteoblasts die within 2 h when deprived of oxygen [60]. Therefore, it has been suggested that tissue-engineered constructs have a maximum size; the acceptable distance from nutrition ranges from 100 mm to 5 mm [61,62]. Due to this phenomenon, ectopic models offer a far more favourable biological environment for implanted cells, because often only few small samples are subcutaneously

G.J. Meijer et al. / Biomaterials 29 (2008) 3053–3061 Table 3 Bone formation after 6 weeks subcutaneous implantation in mice and after 4 months implantation in patients (intra-oral bone defects) Patient Age Gender Alkaline (years) phosphatase staining (ALP)

‘In vivo’ bone formation 6 weeks subcutaneously in mice

Bone formation in human biopsy 4 months after application

5

25

F

þ

Yes

6 7 8 9

19 22 20 45

M M M F

þ þ þ þ

Yes Yes Yes Yes

10

18

F

þ

Yes

Bone in contact with pre-existing bone No Osteoinduction No Bone in contact with pre-existing bone No

implanted, which are in direct contact with the surrounding well vascularised tissues. Therefore, in these models MSCs, convincingly generated bone [2–7,10,13–16]. Obviously, in clinically relevant osseous defects the distance depth exceeds by far maximum of 5 mm. Moreover, cell survival will also be compromised due to the high potassium concentration inside the wound haematoma [61]. It should be emphasised that, in contrast to subcutaneous sites, in osseous defects always an haematoma is formed, thereby inducing an extra hurdle for the survival of the implanted cells [63]. By improving vascularisation, and thus oxygen and nutrient supply, an advancement of ‘in vivo’ cell performance in tissueengineered constructs can be been found [64,65]. The first strategy to stimulate vessel growth is by adding angiogenetic growth factors or endothelial cells to the tissue-engineered construct. Especially, in case of the cell based approach, vessel growth will be stimulated immediately after application [66]. The second method simply bypasses the problems linked to orthotopic bone formation by creating an engineered bone construct in a muscular environment (ectopic bone formation). By this approach the patient serves as his own bioreactor. Warnke et al. [67] reported a successful reconstruction of an extended mandibular discontinuity defect by growth of a custom bone transplant inside the latissimus dorsi muscle of an adult male patient. A prefabricated titanium mesh cage was filled with bone mineral blocks and infiltrated with 7 mg recombinant human bone morphogenetic protein 7 and 20 mL of the patient’s bone marrow. Thus prepared, the transplant was implanted into the latissimus dorsi muscle and 7 weeks later transplanted as a pedicle bone-muscle flap to repair the mandibular defect. The patient involved bore the transplant until his death 15 months later after a cardiac arrest. During this time the graft improved patient’s quality of life [68]. Unfortunately, the external titanium mesh scaffold of the

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mandible replacement fractured near the original mandible stumps, resulting in exposure of parts of the titanium mesh, causing partial infection of the regenerated bone. As an alternative approach to obtain an optimal vascularisation of a construct is suggested to perform a vascular bundle transplantation and subsequently pass the vascular pedunculus through the central hole of a porous calcium hydroxyapatite ceramic block [69,70]. As a third approach, we propose to apply the HMSCs a few days after applying the scaffold. This hypothesis is based on the natural course of bone healing. According to the steps in wound repair, the exudative phase starts immediate after injury; fluid, proteins and cells escape from the damaged vessels and a haematoma forms [71,72]. However, not until the third day during the chronic inflammation phase, blood vessels and fibroblasts proliferate in the fibrin clot, thus forming granulation tissue. Synchronically, from day 3 on, precursor cells as well as mesenchymal cells migrate from the borders of the defect into the granulation tissue to produce bone matrix [73]. In this new approach, the detrimental effect of insufficient oxygen and nutrient supply induced by the haematoma, may be bypassed, because the cultured pre-osteoblasts are injected just a few days after application of the scaffold. At this time point, the new blood vessels are already invading in the haematoma thereby guaranteeing sufficient supply of oxygen and nutrients and thus securing the survival of the implanted cultured HMSCs. A comparable approach is advocated to regenerate heart tissue after infarction with the use of embryonic stem cell-derived cardiomyocytes [74].

5. Conclusions Since the early work of Urist and Friedenstein in the last century, ectopic implantations in rodents are considered as proof of the concept of osteogenesis and osteoinduction. In this study, it is shown that ectopic bone formation in mice is not always predictive for orthotopic bone formation in humans. With respect to biologic environment, there are basic differences between subcutaneously implanted mice compared to osseous defects in humans. To make bone tissue engineering techniques involving cells suitable for reconstructing an osseous defect, the problem of cell death induced by insufficient oxygen and nutrient supply, characteristic for scaffolds with clinical relevant size, must be solved. However, bone formation by implanted cells is feasible, as shown in 1 out of 6 patients. In future research it is important to differentiate between bone formation induced by cells from the border of the osseous defect (osteoconduction) in relation to bone matrix produced by the implanted cells (osteoinduction).

Table 4 Measurement of change in radiographic bone height during 15 months follow-up after reconstruction of the defect Patient number

Location of missing teeth/molar

Amount implanted (ml)

Augmented height (mm)

After 3 months (mm)

After 6 months (mm)

After 9 months (mm)

After 12 months (mm)

After 15 months (mm)

5

4.4 4.5 2.1 2.1 2.1 1.4 1.5 16 1.1 1.2 1.3

1.1

2 2 4 8 4 6 7 8 7 6 7

2 2 4 8 4 6 7 8 7 6 7

1 2 – – – 6 7 7 7 7 7

1 2 3 8 4 6 6 5 7 7 7

1 2 3 8 4 6 6 5 7 7 7

1 2 3 8 4 6 6 – 7 7 7

6 7 8 9

10

0.5 0.5 0.5 2.5

1.5

Note patient 9, location 16; bone height is reduced from 8 to 5 mm indicative for dental implant loss.

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Acknowledgement This research was partially supported by Isotis, Bilthoven, The Netherlands. The principal investigator had full access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.

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