1. Introduction
Postmenopausal osteoporosis is a common skeletal disorder caused by estrogen deficiency. Lack of estrogen leads to the enhancement of bone resorption due to a decreased inhibition of estrogen on both osteoclastogenesis and osteoclast activity, eventually causing decreased bone mass and increased risk for osteoporosis [1]. This disorder is a potential risk factor for biomaterial implants in orthopedics and dental surgery because it could decrease bone formation around implants [2]. Hydroxyapatite (HA), the most commonly used biomaterial in surgery and dentistry, is also susceptible to periprosthetic bone resorption and subsequent loosening in osteopenic ovariectomized animals [3].
Adiponectin (APN), an adipose-derived hormone, has a variety of biological functions and is implicated in insulin resistance, diabetes, obesity, and cardiovascular diseases [4]. Furthermore, results of the recent research studies suggest that APN also plays important roles in bone metabolism. APN might regulate bone formation through autocrine/paracrine and endocrine pathways [5]. APN could increase bone mass by suppressing osteoclast differentiation and activity [6–10]. Although there exists dispute about effects of APN on osteoblast, majority of evidences demonstrate that APN has the potential to activate osteoblasts [6, 7, 11, 12].
Matrigel is a soluble extract of basement membrane proteins derived from Engelbreth–Holm–Swarm (EHS) tumor. Matrigel forms resorbable 3-D structure when warmed to 22–37°C, while it remains in a liquid phase when placed on ice. Matrigel could be used for 3-D cell culture, angiogenesis, and to control the release of soluble growth factors [13].
In this study, Matrigel was designed to develop the controlled release system of APN because the direct local application of APN in vivo would lead to its rapid diffuseness, denature, and degradation in plasma. We have investigated the direct effects of APN released from the system on primary cultures of osteoclastic cells and osteoclast precursor cells in vitro and determined its effects in vivo by improving osteogenesis around HA implant in an ovariectomized rabbit model.
2. Materials and methods
2.1 Materials and treatments
APN (Recombinant Human Adiponectin) and Matrigel used in this study were obtained from R&D systems (Minneapolis, MN, USA) and BD Biosciences (Bedford, MA, USA) respectively. The porous HA bioceramics for bone scaffolds (4mm×5mm×10mm, approximate porosity: 40%, pore size: 5µm–500µm, compressive strength: 15MPa, provided by Research Center for Nano-Biomaterials, Sichuan University) were prepared and randomly divided into 4 groups. The APN was evenly added onto the surface of HA by pippetting the solutions as follows at 4°C respectively: (1) APN+Matrigel+HA group: treated with the mixture of APN and Matrigel (10µg APN+100µl Matrigel per HA); (2) APN+HA group: treated with APN solutions (10µg APN+100µl distilled water per HA); (3) Matrigel+HA group: treated with Matrigel (100µl Matrigel per HA); (4) HA group: treated with 100µl distilled water per HA. Thus the average quantity of APN was approximately 10µg per HA both in APN and APN+Matrigel groups, which was one of the effective volumes described in other studies [10, 14]. After drying under vacuum for 24 h, the samples were packed and stored at −20°C. They were removed from frozen storage where the phase changed from gel to solid (Supplemental Fig. 1), 24h before they were used for the in vitro and in vivo experiment.
2.2 In vitro APN release tests
Elution tests were performed by a high sensitivity direct enzyme-linked immunosorbent assay (ELISA) (BioVendor Laboratory Medicine, Inc., Heidelberg, Germany). The release medium for each measuring time was obtained by placing APN+Matrigel+HA and APN+HA into 1 ml of PBS under continuous agitation. The content of APN release in the medium was measured at 37°C every 24h over 18 days. The total cumulative APN was calculated by integration of each measurement during the release time of the test.
2.3 Culture and activity analysis of mature osteoclasts
2.3.1 Cell Culture and treatment
Rabbit mature osteoclasts were isolated by the method described by Warabi [15]. The cells were treated every 24h with 1 ml release medium from APN+Matrigel+HA, with 1 ml release medium from APN+HA, and with 1 ml PBS. According to the results of our preliminary experiments, there was no difference between Matrigel and PBS regarding the effect on the resorptive activity of rabbit mature osteoclasts (data not shown). Therefore we used PBS as the control medium. The cells were incubated at 37°C, 5%CO2 atmosphere humid incubator, and the culture medium was changed every day. At the 7th day, the cells were tested by tartrate-resistant acid phosphatase (TRAP, TRAP kit, Sigma, USA) and by F-actin Immunofluorescence staining as previously reported [16]. Cells were examined using a scanning laser confocal imaging system (Leica TCS-SP2, Germany).
2.3.2 Bone slice resorption assay
For bone slice resorption assay, sterilized freshly dissected bovine femora discs with 20µm thickness and 5mm diameter were prepared. Mature rabbit osteoclasts were seeded into a 24-well plate separately, with equal doses (2ml) of α-MEM medium containing 1% FBS and a piece of bone disc in each well.
The cells together with the discs were treated every 24h with 1 ml release medium from APN+Matrigel+HA, with 1 ml release medium from APN+HA, and with 1 ml PBS. On the 10th day, the areas of the resorption pits on the bone slices were measured according to the methods previously described [17]. Levels of C-telopeptide of type I collagen (CTx) in the medium were measured using an ELISA kit (Osteometer BioTech, Denmark) according to the manufacturer’s instructions.
2.4 Culture and analysis of osteoclast precursor RAW264.7 cells
2.4.1 Cell Culture and treatment
Osteoclast precursor RAW264.7 cells (ATCC, Manassas, VA USA) were routinely cultured in a 5% CO2 atmosphere at 37°C in α-MEM containing 10% FBS for 3 days. The culturing medium was changed to an α-MEM medium containing 1% FBS and RANKL (50ng/ml), and was treated with 1 ml release medium from the APN+Matrigel group, with 1 ml release medium from the APN group, and with 1 ml PBS. The cells were observed under an inverted phase contrast microscope (IPCM) everyday. The cells were collected on days 3 and 7 for apoptosis analysis and on day 7 for mRNA analysis. 2.4.2 Survival, DNA condensation, and caspase activity assays
The number of viable RAW264.7 cells in three groups were determined by 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT, Invitrogen, Californa, USA) assay as recommended by the manufacturer. The percentage of cell survival was expressed as the ratio of the absorbance of cells at 490nm. DNA condensation and caspase-3/-8/-9 activities were determined using a single-stranded DNA apoptosis ELISA kit (Chemicon, USA) and caspase-3, -8, -9 colorimetric activity assay kits (Chemicon, USA) following the manufacturer’s recommendations.
2.4.2 Real-time RT-PCR for mRNA analysis
Total RNA was isolated from the RAW264.7 cells, using TRIzol reagent (Life Technologies, CA, USA), then reversed and amplified using a First Strand cDNA synthesis kit (Fermentas, Vilnius, Lithuania) and TaKaRa PCR kit (TaKaRa, Tokyo, Japan). The sequences of the primers for amplification were listed as follows, using GAPDH as a control:
TRACP: 5’-GGGAAATGGCCAATGCCAAAGAGA-3’ and 5’-TCGCACAGAGGGATCCATGAAGTT-3’;
Cathepsin K: 5’-AGGCAGCTAAATGCAGAGGGTACA-3’ and 5_-ATGCCGCAGGCGTTGTTCTTATTC-3’;
NFAT-2: 5’-TCTGGTCCATACGAGCTTCG-3’ and 5’-TGGTACTGGCTTCTCTTCCG-3’;
GAPDH: 5’-ATCACTGCCACCCAGAAGAC-3’ and 5’-ATGAGGTCCACCACCCTGTT-3’.
Products were electrophoresed on 1.5% agarose gels, stained with ethidium bromide and visualized using Quantity One software (Bio-Rad, USA). Three independent sets of experiments were performed, each being run three or more times. Quantitative real-time reverse transcription-PCR (qRT-PCR) assay was using a 96-well Real-Time PCR System (7300 Applied Biosystem, USA). The evaluation of relative differences in PCR product amounts was carried out by the comparative cycle threshold (Ct) method, using GAPDH as a control.
2.5 Animal study
2.5.1 Animals
A total of 60 mature female New Zealand rabbits weighing 4.6-0.5 kg were used for the study. Previously described animal handling guidelines were followed [18]. The animal use and care protocol was approved by the Institutional Animal Use and Care Committee.
2.5.2 Treatment
After 7 days of acclimatization, all animals subjected to bilateral ovariectomization (OVX) (n=54) or sham OVX (n=6) surgery. The surgical techniques were described previously [19]. Three months after the surgery, 6 animals were randomly selected in OVX group, and compared with the animals in sham OVX group by micro-CT regarding the microarchitecture alteration of mandibles (data not shown). The left OVX rabbits were randomly divided into 4 groups (12 rabbits per group): APN+Matrigel+HA group, APN+HA group, Matrigel+HA group, and HA group. Each animal received two HA materials treated in the same way. The surgery of implantation is briefly described as follows: After being anesthetized by intraperitoneal injections of xylazine and ketamine (1:3, 1.8 ml/kg), each animal was operated bimandibularly creating a 4mm×5mm×10mm sub-periosteal defect (Supplemental Fig. 2) at inferior border of mandibles by submandibular incision according to the results regarding mandibular mineral density of OVX rabbit [20]. The defected mandible was filled with corresponding HA materials (e.g. APN+Matrigel+HA group received the APN+Matrigel+HA implant, and so on). The incision of skin and soft tissue was closed by a multiple layer wound closure. Postoperatively, the animals were placed in a recovery area with moderate temperature. Prophylactic antibiotics were given IM at the time of operation and for 3 consecutive days postoperatively.
At 4 weeks after implantation, the animals were euthanized. The mandibles with implants were harvested for histological, micro-CT, and biomechanical testing.
2.5.2 Histology and histomorphometry
To decalcify, the mandible samples with HA materials were fixed in 10% neutral buffered formalin for 7 days. After decalcification, the trabecular bone adjacent to the occlusion surface of the HA materials was selected and dehydrated in an ascending series of ethanol, cleared in xylene, and embedded in paraffin.
Ten µm thick tissue sections were performed and mounted on glass slides. Five pieces of histological sections were randomly chosen from each group. After TRAP and H&E staining, each section was observed with 100× magnification by a semi-automated digitizing image analyzer system, consisting of a Nikon ECLIPSE E600 stereomicroscope, a computer-coupled Nikon Digital Camera DXM1200 and NISElements F 2.20 image software. In TRAP staining, 10 images were randomly obtained in one section. The total percentage of TRAP activity was presented as TRAP-positive stained area/total tissue area ×100%, which was consistent with the activity of osteoclasts [21]. In H&E staining sections, the newly formed bone area was restricted to the 0.5 mm area surrounding the HA materials and 10 images were randomly obtained in one section. New bone volume (NBV) was demonstrated as the average percentage of newly formed bone area in the available pore space (bone area/pore area ×100%).
2.5.3 Micro-CT (µ-CT) Scanning
The mandibles with HA materials (n=8/group) were scanned by a µ-CT 80 scanner (Scanco Medical, Bassersdorf, Switzerland) in an axial direction vertical to the long axis of the mandibles. The system was set to 70 kV, 114 mA, 700 ms integration time, 18µm resolution for a detailed qualitative evaluation. After image acquisition, the HA material and mineralized tissue were segmented from each other and from the bone marrow by applying a multilevel threshold procedure. The volume of interest (VOI) included the entire trabecular in 5mm×5mm square (Supplemental Fig. 3) compartment between the cross-sectional planes 1.0 mm proximally and 1.0 mm distally from the HA material. The following microarchitecture parameters were assessed in VOI images: bone volume to total volume ratio (BV/TV), connectivity density (Conn.D), trabecular thickness (Tb.Th), trabecular separation (Tb.Sp) and trabecular number (Tb.N).
2.5.4 Biomechanical testing
The mandible specimens (n=8) were subjected to compressive strength and elastic modulus (E-modulus) biomechanical testing by a commercial material testing system (Instron 4302, Norwood, MA, USA).The specimens were trimmed according to the VOI demonstrated as in µ-CT analysis before the test. The specimens were placed on a sample holder and contacted with the loading press unit at the occlusion surface of the mandibles specimens (Supplemental Fig. 4). The specimens were loaded to failure at a compression speed of 1 mm/min. Ultimate compressive stress and E-modulus was calculated from the stress-strain curve.
2.6 Statistical analysis
The data are presented as mean±SD. One-way analysis of variance (ANOVA), Student–Newman–Keuls (SNK) test, and unpaired Student’s t-test were conducted to analyze differences between the HA group, Matrigel+HA group, APN+HA group, and APN+Matrigel+HA group. Correlation coefficients were calculated to assess the correlation between morphological assessments and the biomechanical assessments. All tests were two-tailed, and all statistical analysis was considered significant when P<0.05.
3. Results
3.1 In vitro release testing
Figure.1 demonstrates the cumulative results of APN released in vitro. The two groups obviously differ in the amount of APN released. The APN+HA group released nearly 90% of the total amount of APN on the first day, and then the APN was scarcely released until day 4 after which there was no further stable and sustainable APN release detected (less than 10ng, and the 24h-release APN on days 5 was 3.5ng). When compared to the APN+HA group, the APN release was obviously prolonged in APN+Matrigel+HA group, and APN was detectable (greater than 10ng) for 16 days (The 24h-release APN on days 16 was 10.2ng). The cumulative level of APN released from the APN+Matrigel+HA increased gradually over time.
Fig. 1.
Cumulative amount of APN released from APN+Matrigel+HA and APN+HA for 18 days.
3.2 The sustained -release APN inhibited resorptive activity of mature osteoclasts
The TRAP and F-actin, two important histochemical markers of osteoclasts, are essential for the resorptive activity of osteoclast [22–24]. To estimate the effects of APN release on the activity of rabbit mature osteoclasts, TRAP and F-actin were tested. The cells in Groups APN+HA and PBS obtained much stronger TRAP positive staining (red) and F-actin ring (red) than that in Group APN+Matrigel+HA (Fig. 2a).
Fig. 2.
(a): Representative images of TRAP staining, F-actin immunofluorescence staining (small white arrow: red F-actin ring), and resorption pits in bone discs (big white arrows). (Original magnification ×200) (b): The areas of the pits formed in Group APN+Matrigel+HA were significantly smaller than those of Group APN+HA or PBS. (c): The CTx in the Group APN+Matrigel+HA was significantly lower than that in APN+HA or PBS. (**: P<0.01)
Rabbit osteoclasts were cultured on bovine bone discs and treated with different concentrations of APN released from their materials. The results showed in Group APN+Matrigel+HA, that the areas of the formed pits were significantly smaller (P<0.01), then between Groups APN+HA and PBS where there was no statistical significance (P>0.05) (Fig.2a and b).
Type I collagen is the main component of bones in mammals. CTx is a specific breakdown product of type I collagen during the process of bone resorption, and is frequently applied as a marker of bone turnover [25]. The cumulative concentrations of CTx in each well were respectively tested. The concentration of CTx in Group APN+Matrigel+HA was significantly lower than in Groups APN+HA and PBS (P<0.01), which was in accord with the results of pit areas. There was no significant difference found between Group APN+HA and PBS (Fig.2c),
According to the results above, it could be inferred that osteoclast activity declined after continuous treatment of APN in Group APN+Matrigel+HA, while the short-term APN treatment in group APN+HA would have slight effect on osteoclasts.
3.3 The sustained-release APN decreased proliferation, increased DNA condensation and caspase-dependent apoptosis, and down-regulates the osteoclastogenic regulators in RAW264.7 cells
At days 3 or 7, RAW264.7 in Group APN+Matrigel+HA exhibited lower MTT readings than that in Groups APN+HA and PBS (P<0.05). At day 3, Group APN+HA showed lower MTT readings than that in Group PBS (P<0.05) (Fig. 3a and b). The MTT test showed that the proliferation of RAW264.7 cells induced by RANKL was inhibited by APN treatment.
Fig. 3.
(a): Representative images of RAW264.7 cells in Groups APN+Matrigel+HA, APN+HA, and PBS at 7 days (IPCM, original magnification ×200). (b): The proliferation of RAW264.7 cells induced by RANKL in each group was determined by MTT assay. The cells in Group APN+Matrigel+HA exhibited lower MTT readings than that in Group APN+HA and PBS at 3 or 7 days. Group APN+HA showed lower MTT readings than that in Group PBS only at 3 days. (c): The DNA condensation in Group APN+Matrigel+HA and APN+HA was higher than Group PBS at 3 days. Group A had higher DNA condensation contrast to Group APN+HA or PBS at 7 days. (d–f): The activity levels of caspase-3/-8/-9 were all higher in Group APN+Matrigel+HA than those in Group APN+HA and PBS at 3 or 7 days. (*: P<0.05, **: P<0.01)
To analyze whether the sustained-release APN treatment increases apoptosis of RAW264.7 cells, the DNA condensation and caspase-3/-8/-9 activities were tested. On the 3rd day, the DNA condensation in Groups APN+Matrigel+HA and APN+HA was higher than in Group PBS, and on the 7th day, Group APN+Matrigel+HA had a higher DNA condensation than Groups APN+HA or PBS (P<0.05) (Fig. 3c). Similarly, the results of caspase-3/-8/-9 activity tests demonstrated that the activity levels of the three caspases were all higher in Group APN+Matrigel+HA than those in Groups APN+HA or PBS at days 3 or 7 (P<0.05) (Fig. 3d–f). Thus, the sustained-release APN increased caspase-dependent apoptosis in RAW264.7 cells treated by RANKL.
NFAT2 is the master regulator of osteoclastogenesis[26], while TRACP and cathepsin K are two important osteoclastic differentiation markers[27, 28]. The mRNA levels of these three markers were monitored as osteoclastic differentiation markers. The sustained-release APN down-regulated the expression of TRACP, cathepsin K, and NFAT2 of RAW264.7 cells treated with RANKL. However, no significant differences could be found between Groups B and C (P>0.05) (Fig.4). These results proved that continuous APN treatment inhibited osteoclastic differentiation, while the short-term APN treatment did not.
Fig. 4.
(a): RT-PCR results of TRACP, cathepsin K and NFAT2 in Groups APN+Matrigel+HA, APN+HA, and PBS at day 7. (b) The mRNA levels of TRACP, cathepsin K, and NFAT2 of RAW264.7 cells treated with RANKL were monitored by Real-time RT-PCR. The cells in Group APN+Matrigel+HA had lower expression levels of these three markers in contrast to Groups APN+HA or PBS at 7 days. (*: P<0.05, **: P<0.01)
3.4 Histomorphometry analysis
All rabbits completed the 4 week observation time uneventfully. Histomorphometrically, the APN+Matrigel+HA group had weaker staining for TRAP activity than the other three groups (P<0.05) (Fig.5a and b). Consistent with this, the APN+Matrigel+HA group had the highest level of NBV, which was statistically different from the other groups (P<0.05) (Fig.5a and c). There was no difference among APN+HA, Matrigel+HA, and HA groups (P>0.05). It could be inferred that the effect of APN on osteoclast activity and levels of NBV around the implants was influenced by persistence of APN treatment. No significant differences were found between the Matrigel+HA and HA groups, which demonstrated that Matrigel had no obvious effect on the peri-implant osteogenesis.
Fig. 5.
(a): Representative images of TRAP and H&E staining (black arrow: TRAP-positive stained area; white arrow: newly formed bone area; original magnification ×100). (b) and (c): APN+Matrigel+HA group demonstrated the weaker staining for TRAP activity and the highest levels of NBV than the other three group. There was no difference among the APN+HA, Matrigel+HA, and HA groups. (*: P<0.05)
3.5 µ-CT analysis
The µ-CT analysis demonstrated that there was a significant difference between the APN+Matrigel+HA group and the other three groups (P<0.05). The APN+Matrigel+HA group had the highest BV/TV, Conn.D, Tb.Th, and Tb.N., as well as lowest Tb.Sp. Compared with the HA group, the APN+Matrigel+HA group had an increased BV/TV by 40.6%, Conn.D by 54.9%, Tb.Th by 41.9%, Tb.N by 41.1%, and decreased Tb.Sp by 41.2%. Compared with the APN+HA group, the APN+Matrigel+HA group also had an increased BV/TV by 35.3%, Conn.D by 41.1%, Tb.Th by 32.4%, Tb.N by 30.3%, and decreased Tb.Sp by 38.9% (Fig. 6a–f). Sustained-release APN exhibited improvement to osteogenesis around the HA materials. No differences could be found between APN+HA and HA group (P>0.05). It could be referred that short-term APN treatment could not improve the peri-implant osteogenesis. Matrigel+HA showed similar results when contrasting to APN+HA and HA group (P>0.05). It was proved that Matrigel had no obvious effect on osteogenesis around HA materials.
Fig. 6.
(a): Representative images of 2-D µ-CT scanning. (b–f): Microarchitecture parameters measured by µ-CT. APN+Matrigel+HA group had the highest BV/TV, Conn.D, Tb.Th, Tb.N., and the lowest Tb.Sp. There was no difference among the APN+HA, Matrigel+HA, and HA groups. (*: P<0.05)
3.6 Biomechanical testing
Biomechanical tests also demonstrated there was a significant difference between the APN+Matrigel+HA group and other three groups (P<0.01) (Table.1). Specimens from the APN+Matrigel+HA group revealed the highest values of both compressive strength (P<0.005) and E-modulus (P<0.01). Sustained-release APN induced a marked increase of ultimate compressive stress by approximately 1.6-fold, E-modulus by approximately 0.85-fold, compared with the APN+HA, Matrigel+HA, or HA groups respectively. No difference could be found between the APN+HA, Matrigel+HA, and HA groups (P>0.05).
Table 1.
Compressive strength and E-modulus in biomechanical testing.
| Groups | Ultimate compressive stress (Mpa) |
E-modulus (Mpa) |
|---|---|---|
| APN+Matrigel+HA | 8.34±0.171 | 38.74±4.17 |
| APN+HA | 3.04±0.093** | 20.37±2.85* |
| Matrigel+HA | 3.11±0.138** | 22.04±3.12* |
| HA | 2.98±0.052** | 20.12±1.28* |
Data values are expressed as mean±SD,
P<0.01 vs. APN+Matrigel+HA group,
P<0.005 vs. APN+Matrigel+HA group
Furthermore, biomechanical and µ-CT parameters demonstrated significant correlation coefficients (P<0.05) (Table.2). The order of these coefficients suggests that the strongest relationship of the maximum compressive stress was with BV/TV. It could be inferred that sustained-release APN could improve the biomechanical characteristics by promoting peri-implant osteogenesis.
Table 2.
Correlation coefficients between biomechanical and µ-CT parameters.
| Ultimate compressive stress (Mpa) |
E-modulus (Mpa) |
|
|---|---|---|
| BV/TV | 0.87 | 0.85 |
| Conn.D | 0.62 | 0.60 |
| Tb.Th | 0.74 | 0.74 |
| Tb.Sp | −0.61 | −0.81 |
| Tb.N | 0.59 | 0.66 |
All coefficients were significant at P<0.05.
4. Discussion
In the present study, we investigated the effect of sustained-release APN on osteoclasts in vitro and osteogenesis around hydroxyapatite implants in vivo, using Matrigel as the controlled medium. The major finding of the present study was that the sustained-release APN could suppress the oseoclastic activity and improve the peri-implant osteogenesis in OVX rabbits. The analysis of the mechanism of this action revealed that the sustained-release APN might promote peri-implant osteogenesis through bone remodeling by reducing the differentiation and bone-resorption activity of osteoclasts, and enhancing the apoptosis of osteoclasts.
OVX animal model is the most commonly used and extensively studied experimental animal model of postmenopausal osteoporosis. In OVX-induced osteoporosis animals, bone healing around HA implants was delayed compared with normal animals due to the highly activated osteoclasts [2,3,29]. Some methods were reported to improve dental implant osseointegration in such condition, including individual or synergistical treatment of bisphosphonate, strontium ranelate, basic fibroblast growth factor, and so on [30–32]. But few studies have reported how to promote the bone defect restoration by HA scaffolds in OVX or osteoporotic aniamals [33].
APN is highly and specifically expressed in differentiated adipocytes and is abundantly present in plasma, and reduced level of APN closely relates to the pathophysiology of insulin resistance and Type 2 diabetes mellitus (T2DM) [34]. Furthermore, APN is also an important negative regulator in hematopoiesis and immune system homeostasis, by performing anti-inflammatory actions through its inhibitory functions including suppression of IL6 and induction of IL10, suppression of macrophages, and IL6, and suppression of NF kappa B signaling and ERK1/2 activity [35–37] .
Recent studies have revealed that APN also plays an important role in bone metabolism. Most studies have reported that APN significantly inhibits M-CSF- and RANKL-induced osteoclast differentiation and function [6–10], with the exception of one group of researchers who thought that APN had no direct effect on osteoclasts [38]. Alternatively, results derived from studying the effects of APN on osteoblast have also shown discrepancies, but the majority cumulative evidence has demonstrated that APN has the potential to activate osteoblasts [6, 7, 11, 12].
In the current study, we used a gel of Matrigel incorporating APN for improvement of peri-implant osteogenesis in osteopenic rabbits. Matrigel could resemble the 3-D complex extracellular environment found in many tissues; it has been used as a substrate for cell culture or a carrier for soluble growth factors [13, 39, 40]. Though Matrigel has also been shown to be highly effective in hastening revascularization [41, 42], there is no convincing evidence revealing whether Matrigel could increase or decrease osteogenesis. We performed preliminary experiments, where no difference was found between Matrigel and PBS regarding the effect on the rabbit bone marrow derived stroma cells (BMSCs), osteoblasts or mature osteoclasts (data not shown). The results of our in vivo study also proved Matrigel has no obvious intrinsic effects for the promotion of bone regeneration by itself. Matrigel mainly acted as a controlled release medium in this study. Matrigel could not only prolong the duration of release of APN to achieve the long-term persistence, but also protected this therapeutic cytokine against the enzymatic degradation in vivo to maintain the bioactive effects [33, 41].
In this study, we observed that the effect of APN on osteoclast differentiation and function was closely related to the treatment duration. In the sustained release group, the treatment duration of APN was more than 16 days, and osteoclast proliferation and resorptive activity were obviously suppressed. The suppression resulted from, at least in part, by the down-regulation of the osteoclastogenic gene and the up-regulation of osteoclastic apoptosis according to our experimental results. While in the APN-HA group, no similar suppression function could be found.
Our results derived from the in vitro studies were supported by the in vivo study. The TRAP staining revealed that osteoclastic resorptive activity was significantly suppressed around the HA materials in the APN+Matrigel+HA group. Accordingly, this group also showed a modest improvement in the bone density and trabeculae microarchitecture around implants when compared with other the groups. Biomechanical testing further demonstrated that the sustained-release APN could increase the biomechanical property of the mandibular defect repair. µ-CT and biomechanical parameters were significantly correlated. These consistent results supported the beneficial effect of the sustained-release APN on osteogenesis around HA implants.
Although the current study revealed APN might act by reducing the differentiation and bone-resorption activity of osteoclasts, and enhancing the apoptosis of osteoclasts, other mechanism should not be ignored, given that APN has multiple functions in anti-inflammation [43] and bone metabolism [7]. Specially, BMSCs or/and osteoblasts might play an important role to assist APN to improve peri-implant osteogenesis, which need further studies.
Using rabbits as experimental animals we were able to obtain enough bone volume for feasibility and accuracy of the experiment. Although OVX rabbits have certain advantages for the study, including a much faster bone turnover and a shorter time to reach skeletal maturity compared with rodents [20, 44], future studies would be greatly beneficial to perform using the OVX rat as a pre-clinical animal model to confirm the actions of APN on peri-implant osteogenesis. In addition, it had no obvious effect on the peri-implant osteogenesis according to the results of this study, but special attention should be paid to Matrigel in vivo, since it is actually extracellular matrix proteins derived from mouse, and could accelerate revascularization. The degradation and longevity of Matrigel in vivo is also necessary to be determined.
5. Conclusions
The results of this study demonstrated that long-term persistence of APN could promote peri-implant osteogenesis in OVX rabbits. The mechanism of this action is, at least in part, through suppressing the differentiation and bone-resorption activity of osteoclasts, and enhancing the apoptosis of osteoclasts. It is suggested that sustained-release APN may be an effective strategy for improvement on restoration of bone defect by HA materials under an osteoporotic condition in which osteoclast is highly activated.
Supplementary Material
Scanning Electron Microscope micrographs of surface morphology of HA materials without Matrigel (A) or with Matrigel (B) (at 24°C). (Original magnification, ×100)
A radiography of the bone defect (white arrow) created at the inferior border of rabbit mandible.
The VOI of µ-CT analysis (white square). After be segmented from HA material and bone marrow by a multilevel thresholding procedure, mineralized tissue was used for microarchitecture parameter analysis.
Schematic drawing of biomechanical testing. (F: compressive force)
Acknowledgements
This study was supported by grants from the financial support of the National Science Foundation of China (30973346), Program for New Century Excellent Talents in University (NCET-10-0597) and NIH grants DE16710 and DE21464 to JC.
Footnotes
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Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.
Supplementary Materials
Scanning Electron Microscope micrographs of surface morphology of HA materials without Matrigel (A) or with Matrigel (B) (at 24°C). (Original magnification, ×100)
A radiography of the bone defect (white arrow) created at the inferior border of rabbit mandible.
The VOI of µ-CT analysis (white square). After be segmented from HA material and bone marrow by a multilevel thresholding procedure, mineralized tissue was used for microarchitecture parameter analysis.
Schematic drawing of biomechanical testing. (F: compressive force)