Retention of removable dentures is often a problem for edentulous patients with severe maxillary or mandibular atrophy, a common problem in the aged population. Installation of dental implants can help these patients by providing better support for and retention of their dental prosthesis. Placing dental implants requires sufficient jaw bone volume, which is often not available, especially in the edentulous distal maxillary area of severely atrophied maxillae. In these cases, the bone volume must be increased by augmentation prior to dental implant placement.
Maxillary sinus floor elevation (MSFE) or augmentation has been introduced to dentistry in the mid-1970s (modified by Tatum in 1986) [1
] and has become part of pre-prosthetic surgery. In this surgical procedure, the distal area of the maxilla is augmented by transplanting bone or bone substitutes to the bottom of the maxillary sinus. The maxillary sinus floor elevation model is unique by allowing histological examination of biopsies obtained during the preparation for dental implant placement using a hollow trephine bur.
In dentistry, autologous bone grafting is still the “gold standard” for bone augmentation. Autografting, however, has an important disadvantage, i.e., trabecular bone needs to be harvested from elsewhere in the skeleton, usually from the lateral mandible, the chin, or the iliac crest, and applied to the jaw defect. This means another operation site, including the risk of hospitalization, causing donor site morbidity, and potential complications, such as post-operative infections, and functional defects [2
]. Therefore, alternative bone substitute materials have been evaluated. Calcium phosphates, such as hydroxyapatite (HA), β-tricalcium phosphate (β-TCP), and a combination of HA/β-TCP, are often used since they do not evoke adverse cellular reactions and, in time, the material is either replaced by bone or integrated into the body, depending on the degradation properties [3
]. However, these materials have limitations due to the slow speed of osteoconduction when compared to the properties of autologous bone [6
]. The time required is strongly determined by the time that osteogenic cells take to grow into the augmentation space and would potentially be shortened if bone regeneration-competent cells (mesenchymal stem cells; MSCs) are mixed with the bone substitute. The calcium phosphate carrier has controlled 3D properties allowing immediate colonization by MSCs of the entire volume of the ceramics, and in depth revascularization [9
Endothelial cells are lining blood vessels and allow the formation of new blood capillaries by the sprouting of an existing small vessel, a phenomenon called angiogenesis. This process allows tissue growth and repair by extending and remodeling the network of blood vessels [11
]. In contrast to small blood vessels, composed of endothelial cells surrounded by a basal lamina and loosely covered by single pericytes, larger vessels are coated with multiple layers of smooth-muscle cells and elastic and collagenous fibers [6
]. These are composed by the endothelium, a thin layer of endothelial cells, separated from the surrounding outer layers by a basal lamina. The amounts of connective tissue and smooth muscle in the vessel wall vary according to the vessel’s diameter and function, but the endothelial lining is always present. Pericytes have been associated mainly with stabilization and hemodynamic processes of blood vessels. Their functions are, however, much more diverse than traditionally thought. They can sense angiogenic stimuli, guide sprouting tubes, elicit endothelial survival functions, and even exhibit macrophage-like activities which make them crucial cells in the process of tissue repair and remodeling [13
Poor angiogenesis is a common and vital barrier to tissue regeneration. Regenerating tissue over 200 µm exceeds the capacity of nutrient supply and waste removal from the tissue and, therefore, requires an intimate supply of vascular networks [14
]. This has led to the use of angiogenic growth factors and/or transplantation of proangiogenic cells, such as endothelial progenitor cells (EPCs), in combination with scaffolds. The use of angiogenic growth factors and/or transplantation of these proangiogenic cells only, however, also has disadvantages, since perivascular cells, including mural cells, are obligatory for the formation of native, multilayered mature microvessels [15
]. The potential of MSCs to stimulate angiogenesis holds interesting promises to the field of tissue engineering.
Adipose tissue may be easily obtained from patients using liposuction or oral surgical procedures [16
]. Adipose tissue represents a promising source of MSCs, as liposuction can be performed with minimal patient discomfort and yields higher numbers of MSCs than bone marrow, which could avoid costly cell expansion to obtain a number of cells high enough for clinical use [17
]. Risks associated with cell culturing such as pathogen contamination, spontaneous transformation and loss of proliferation and differentiation potential are thus minimized [20
]. The stromal vascular fraction (SVF), obtained immediately after digestion of adipose tissue with collagenase and centrifugation to separate the floating adipocytes, is highly heterogeneous and contains many cell subsets, including native adipose stem cells (ASCs), mature endothelial cells, and haematopoietic cells [19
]. SVF also contains macrophages, which secrete a multitude of vascular growth factors and cytokines [23
]. The ASCs in SVF have been shown to attach, proliferate, and osteogenically differentiate on calcium phosphate scaffolds [24
], and secrete a high number of growth factors [25
]. ASCs have not only shown osteogenic potential in vivo [26
], but also demonstrated angiogenic potential crucial for bone tissue engineering applications in mice [28
]. This supports in vitro observations that ASCs in SVF secrete a variety of angiogenic and anti-apoptotic growth factors [29
], and that SVF is highly enriched with CD34+CD45− cells. The CD34+ cells are capable of stimulating angiogenesis, and are involved in neovascularization processes that facilitate healing of ischemic tissues in mouse models [30
]. However, whether SVF/ASCs are also effective in stimulating vascularization in humans has not been unequivocally shown so far.
In an earlier performed clinical phase I study [27
], employing our one-step surgical procedure [18
] in patients undergoing maxillary sinus floor elevation, we isolated SVF, and re-implanted them intraoperatively into the patient again. This study successfully showed feasibility, safety, and potential efficacy of using bioactive implants consisting of calcium phosphate carriers seeded with freshly isolated SVF containing ASCs. Since we hypothesized that the SVF will positively contribute not only to bone formation, but likely also to vascularization, the current study aimed to evaluate vascularization in relation to bone formation potential of SVF in biopsies from the previous study obtained after six months (when dental implant placement occurred). For this evaluation, a recently developed immunohistochemical staining technique for methyl methacrylate (MMA) polymer resin embedded bone biopsies [31
] was applied.
2. Materials and Methods
2.1. Clinical Study Outline
This angiogenic study is an extension of a phase I/IIa clinical trial study we reported on before [27
], in which safety and potential efficacy of maxillary sinus floor bone augmentation using calcium phosphate bone substitutes and a freshly-isolated adipose stem cell preparation, termed the stromal vascular fraction (SVF), was evaluated in a one-step surgical procedure in 10 partially-edentulous patients requiring dental implants for prosthetic rehabilitation. The clinical study was registered in the Netherlands Trial Registry (NTR4408), and complied with the principles of the Declaration of Helsinki of 1975, revised in 2008. All protocols were approved by the medical ethics committee (IRB) of the VU University Medical Center Amsterdam, as well as the central committee on research involving human subjects (CCMO, The Hague, The Netherlands; Dossier number: NL29581.000.09; EudraCT-number: 2009-015562-62). All patients signed a written informed consent before participation in the study. For detailed inclusion and exclusion criteria, procedures and assessments according to the study protocol of our previously performed phase I trial, one is referred to our previous report [27
]. The demographic data of our patient group is summarized in Table 1
A graphic visualization of the surgical protocol is depicted in Figure 1
. Briefly, the surgery started by collecting >125 mL of adipose tissue using a syringe-based lipoaspiration. The lipoaspirate (Figure 1
a) was subsequently transported to a special stem cell laboratory within the VU University Medical Center Amsterdam operation complex. Within the stem cell laboratory, the adipose tissue was processed with the CE marked Celution device (Cytori Therapeutics, Inc., San Diego, CA, USA) (Figure 1
b) to obtain SVF. Viability and cell number was determined in triplicate with a Nucleocounter NC-100 (ChemoMetec A/S, Allerød, Denmark) according to the manufacturer’s protocol. The release criterion was set at ≥70% viability. For implantation cells were seeded in Ringer’s lactate solution in a concentration of 107
nucleated SVF cells (±2 × 105
ASC-like cells)/g calcium phosphate carrier (Figure 1
c). Calcium phosphate carriers consisted of 100% Ceros®
β-TCP with 60% porosity and granule size of 0.7–1.4 mm (Thommen Medical, Grenchen, Switzerland) or Straumann®
Bone Ceramic biphasic calcium phosphate (BCP), consisting of 60% hydroxyapatite (HA) and 40% β-tricalcium phosphate (β-TCP) with 90% porosity and granule size of 0.5–1.0 mm (Straumann AG, Basel, Switzerland). After allowing attachment of the cells for 30 min and subsequent washing with Ringer’s lactate solution to remove unattached cells, the carriers were implanted using a standard maxillary sinus floor elevation procedure according to the lateral “top hinge trap door” procedure of Tatum [1
] (Figure 1
d–f). In the case of a bilateral “split-mouth” design treatment, one side was implanted with the cell-seeded carrier, while the other (control) side was implanted with carriers undergoing the same seeding procedure, but with vehicle (Ringer’s lactate solution) only.
Bone biopsies were obtained during dental implant surgery after a six month healing period, using hollow trephine drills with an external diameter of 3.5 mm (Straumann AG, Basel, Switzerland) under local anesthesia prior to dental implant placements (Figure 1
g). After a three month osseointegration period the suprastructures were manufactured, and placed by the patient’s dentist.
2.2. Biopsy Processing and Evaluation
The biopsies were fixed in 4% formaldehyde solution (Klinipath BV, Duiven, The Netherlands) at 4°C for 24 h, removed from the drill, transferred to 70% ethanol, and stored until use for histomorphometrical analysis (Figure 2
), as described below. For a valid, uniform comparison of the biopsies taken from the sides that were treated with calcium phosphate only and the biopsies taken from the sides augmented with calcium phosphate and ASCs, a selection was made by two independent experienced observers. The biopsies taken from implant sides outside the augmented maxillary sinus (mainly implant position 14 and/or 24) were excluded from analysis. Per patient, one biopsy from each side was selected in the middle of the grafted area to exclude the effect of surrounding bone containing mechanically-loaded dental elements and bone near the nasal wall of the maxillary sinus. Using these selection criteria, a total of 16 selected biopsies (six from the control side, 10 from the study side) from 10 patients were studied to analyze the blood vessel formation and bone formation (Table 1
, bold numbers). In addition, one sample from a transilical biopsy was used as control for the immunohistochemical analysis.
2.3. Histology and Histomorphometry
After dehydration in descending alcohol series, the bone specimens were embedded without prior decalcification in low temperature polymerizing methylmethacrylate (MMA, Merck Schuchardt OHG, Hohenbrunn, Germany). Longitudinal sections of 5 μm thickness were prepared using a Jung K microtome (R. Jung, Heidelberg, Germany). Midsagittal histological sections of each biopsy were stained with Goldner’s Trichome method [6
], in order to distinguish mineralized bone tissue (green) and unmineralized osteoid (red). The histological sections were divided into regions of interest (ROI) of 1 mm2
for blinded histomorphometrical analysis, as previously described [32
]. Depending on the length of the biopsy, the number of ROIs ranged from 9–15. The digital images of the scanned biopsies were analyzed, starting from the caudal side of the biopsy, and continuing towards the cranial side. This method allowed to compare similar ROIs for all biopsies (with and without stem cells) with respect to the bone regeneration and blood vessel formation in the augmented maxillary sinus. For each ROI, the bone volume (BV) was calculated as a percentage of the total tissue volume (TV), as previously described [33
]. This analysis was performed by two independent blinded observations.
For each separate area of interest, the histomorphometrical measurements were performed with a computer using an electronic stage table and a Leica DC 200 digital camera (Leica, Wetzlar, Germany). The computer software used was Leica QWin© (Leica Microsystems Image Solutions, Rijswijk, The Netherlands). Digital images of the sections were acquired at 100× magnification. Consecutive ROI of 1 mm2
each were defined and numbered throughout the whole biopsy. The transition zone (TZ) indicates the first ROI where graft material was observed when analyzing from the caudal to the cranial side of the biopsy. Because the biopsies analyzed had different lengths, we decided to define them in three regions after the transition zone (TZ) between the native bone or caudal area and the scaffold area towards cranial. The first two ROIs on the right of the transition zone were defined as region I, the two or three ROIs in the middle (even or odd numbers) as region II, and the two rightmost ROIs as region III (Figure 2
Data from the residual native bone part of the biopsy next to the transition zone and for each area from the sinus floor towards the cranial side of the biopsy was analyzed separately (Figure 2
). Similar to this method we have already been able to compare similar areas of interest for the two sides (control and test side) with respect to the bone regeneration performance indicated by the amount of osteoid and bone formed, and volume of remaining graft material [32
Blood vessel numbers, taking into account the blood vessel size, were determined as mean value of two separate blinded counts. Blood vessel size was calculated as the total blood vessel area expressed in µm2. According to their size, blood vessels were divided into small (0–400 µm2) or large vessels (>400 µm2).
To quantify microvessel density, immunohistochemical staining for CD34, a marker of endothelial cells, as well as stem cells, such as endothelial progenitor stem cells and hematopoietic stem cells, was performed [34
]. The expression of smooth muscle actin (SMA), a marker of smooth muscle cells as well as pericytes, was also analyzed by immunohistochemistry [35
]. The presence of pericytes and smooth muscle cells surrounding blood vessels has been described as a structural parameter indicative of vascular maturity.
The MMA in the sections was removed by immersing them in xylene/chloroform (Merck, Darmstadt, Germany) for 30 min at room temperature followed by two rinses with xylene. Bone sections were rehydrated in graded alcohol solutions. To block endogenous peroxidase when using the avidin biotin peroxidase complex the hydrated specimens were transferred to 3% (v/v) hydrogen peroxide in methanol for 15 min. In a set of pilot experiments, it was deduced that optimal CD34 staining results were achieved with 5 µg/mL proteinase K pretreatment for 10 min (Invitrogen, Carlsbad, CA, USA), while SMA detection was best without proteinase K predigestion. Non-specific binding of immunoglobulin G was blocked by incubation with 5% (v/v) normal serum (as appropriate for each antibody) for 60 min with 0.1% bovine serum albumin (Sigma, St Louis, MI, USA). The sections were then incubated with the primary antibodies at optimal dilutions for 2 h at room temperature (SMA Monoclonal Mouse Anti-Human Smooth Muscle Actin Clone 1A4 1:50, Dako, Carpinteria, MI, USA) and overnight incubation at 4 °C in a humidified chamber (CD34 Monoclonal Mouse Anti-Human CD34 Class II Clone QBEnd-10 1:20, Dako). Sections were rinsed in phosphate-buffered saline and incubated at room temperature with an horseradish peroxidase (HRP)-labelled polymer conjugated with secondary antibody (Envision Kit, Dako, Santa Clara, CA, USA), for 0.5 h at room temperature before detection using aminoethyl carbazole (AEC) (Invitrogen, Carlsbad, CA, USA) staining as recommended by the manufacturer’s protocol. Sections were counterstained with haematoxylin (Merck, Schuchardt OHG, Hohenbrunn, Germany). Sclerostin (hSOS, Dako, Santa Clara, CA, USA) was used as positive control. Negative controls (without primary or secondary antibodies, or both) were also performed for all antibodies tested.
The determination of the number of blood vessels was performed as previously described [37
]. The number of blood vessels was expressed per area of soft connective tissue (mm2
]. Blood vessel size was calculated as described above.
Data are presented as mean ± standard deviation (SD). Data analysis and statistical analysis were performed using GraphPad Prism 5 software (GraphPad Software, La Jolla, CA, USA) and IBM SPSS 23 statistical software (CircleCI, San Francisco, CA, USA). Selected biopsies from all treated patients (with vs. without stem cells) were compared between the β-TCP and BCP groups.
A paired Wilcoxon signed rank test was performed to assess whether bone volume and blood vessel number were higher at the study sides compared to control sides for each material. An unpaired nonparametric Mann Whitney U test was performed to test differences between β-TCP and BCP in control and study samples. Statistical significance was considered if p-values were <0.05.
In bone tissue engineering adequate vascularization is crucial for timely and adequate transport of nutrients and waste removal, and the provision of progenitor cells for tissue remodeling and repair. It is widely agreed that vascularization and bone formation are highly linked, and that vascularization precedes osteogenesis during both embryonic development and adult bone healing [38
]. Since we hypothesized that supplementation of bone substitutes with SVF, containing adipose stem cells, will positively contribute not only to bone formation, as shown previously [27
], but likely also to vascularization, the current study aimed to evaluate vascularization (number and size of blood vessels) in relation to bone formation potential of SVF.
Histomorphometric analysis and quantitative assessments of CD34- and SMA-immunostained blood vessels showed: (i) a clear trend towards increased bone formation in the SVF-supplemented group vs. the control group, in particular in the most cranial part (region III) of the biopsies, which was significantly higher in the β-TCP vs. the BCP group; (ii) comparable patterns of angiogenic and bone formation levels; (iii) comparable total blood vessel numbers obtained from histomorphometric and immunohistochemical analyses; (iv) SVF significantly promoted CD34+ large vessel formation in region I in β-TCP, but not BCP; (v) in the BCP group supplemented with SVF, region II displayed considerably higher CD34+ small and large vessel numbers; and (vi) in the absence of SVF the β-TCP, but not the BCP scaffolds, promoted both small and large vessel numbers in region II.
Our data analysis of the events occurring in the graft area using a division in regions provides important additional insight in course of events and local differences which would otherwise have been masked in a more global evaluation of the total graft area as a whole. The striking observation of the far more active bone formation observed at the cranial side of the biopsies in the case of SVF supplementation (7/10 biopsies) compared to the control side (1/6), as reported before [27
], appears to match our current finding of higher blood vessel counts and the presence of more mature vessels (amongst others characterized by SMA+ staining) in these areas of active bone formation. The strong angiogenic potential of SVF has already been reported in numerous other studies [26
]. The current findings in the six-month human model also adds to our earlier findings in previous preclinical large animal studies of our group in which we found that already after one month, SVF generates larger, more mature vessels compared to non-supplemented scaffolds [40
]. The diameter of the blood vessels (mean diameter 30 µm) is well above the size of capillaries (5–10 µm) and can, therefore, be considered sufficient for bone formation [40
]. Unfortunately, our study setup did not allow conclusions on blood vessel orientation, for it would have been interesting to evaluate whether the vessels would align in the principal loading direction.
Future studies with inclusion of a higher number of patients might reveal significant differences between groups where significance could not be reached due to the relatively low number of patients included in the current study. It would be interesting to also determine the orientation of the blood vessels based on an analysis of the blood vessel cross-sectional area, to elucidate if the orientation of blood vessels may be different in the scaffold area of stem cell-treated patients compared to controls.