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Physical, microarchitectural, and biocompatible properties of natural latex extracted from Hevea brasiliensis were initially determined using animal models and are now being studied in humans (Frade et al., 2001). Initial studies have found that latex is an innovative biomaterial with excellent results in healing chronic ulcers and reconstituting perforated esophagi and timpani (Oliveira et al., 2003). Latex has also been used for reconstructing temporal muscle fascia (Oliveira et al., 2003) and as arterial prosthesis in animal models, healing of ocular conjunctiva and neoangiogenesis in rabbits (Pinho et al., 2004). The biocompatibility of latex has also been studied for repairing bone defects in dental alveoli (Balabanian et al., 2006).
Recombinant human bone morphogenetic protein-2 (rhBMP-2) is commercially available and presents good osteoinductive capabilities (Wang et al., 2009). In vivo (Fujimura et al., 1995) and in vitro (Zhao et al., 2009) studies have found that rhBMP-2 rapidly diffuses from the application site when deposited in solution, diminishing the osteoinductive effect. Recent studies have demonstrated that the application of larger amounts of rhBMP-2 directly into the tissues is not as effective for osteogenesis induction because of fast protein metabolism (Wang et al., 1990). Sustained lower quantities seem to produce better results (Lee et al., 1994; Bostrom et al., 1995; Issa et al., 2008).
The presence of a carrier material influences newly formed bone volume and diminishes the total amount of protein needed to induce bone formation (Miyazaki et al., 2009). Collagen is a common carrier used with rhBMPs. It is a biocompatible material and effective for slow release of rhBMP-2, regardless of the implantation site (Toriumi et al., 1990; Kirker-Head et al., 1995; Sigurdsson et al., 1995; Sigurdsson et al., 1996). Collagen offers cells a permissive substrate for union and differentiation (Sampath and Reddi, 1981; Reddi, 1992) and serves as a migration barrier of undesirable cells to the surgical site (Thoma et al., 2009). Collagen is the main component of the extracellular matrix and influences cell biology by sensitizing cell surface receptors, interacting with growth factors (BMPs), and providing an interactive environment (Sampath and Reddi, 1981; Paralkar et al., 1990; Reddi, 1992).
This study used monoolein (glyceryl monooleate) as the carrier for rhBMP-2. Monoolein is biodegradable, biocompatible, degraded by sterase, and end product of lipid digestion (Norling et al., 1992; Stoltze, 1995; D'Antona et al., 2000). It has been used as a drug carrier in various pharmaceutical forms and routes of administration (Shah et al., 2001), including semi-solid matrices, subcutaneous and intramuscular implants (Malonne et al., 2000), intravenous solutions (Lee and Kellaway, 2000a, 2000b), gels for intranasal application (Ramanathan et al., 1998), and as carrier for rhBMP-2 release (Issa et al., 2008). Bioadhesive properties of monoolein also make it beneficial for transmucosal release (Geraghty et al., 1997; Lee et al., 2001) and stability. The aim of this study was to evaluate the in vivo osteogenic potential of two proteins, rhBMP-2 and P-1, a natural latex extracted from Hevea brasiliensis, and to determine the effects of combined treatment with a collagen gelatin on bone defects.
MATERIALS AND METHODS
Natural Latex Collection
Latex was collected from several clones of the Hevea brasiliensis through half-spiral shaped incisions in the trees bark. Natural latex extracted from Hevea brasiliensis used in this study was mixed with 0.5%–2.0% ammonium hydroxide as preservative and to retain a liquid state.
Purification of the Natural Latex
The ammonium latex was diluted in 2.2% acetic acid (1:2, v/v) at the Medical School of the University of São Paulo. The diluted latex was then homogenized and left at room temperature for 30 minutes. Serum was then separated from the rubber and submitted to chromatographic separation using ionic exchange chromatography with DEAE-Celluloses. Serum was diluted in distilled water (1:1 v/v) and pH adjusted to 9.0. This material was applied to the chromatographic column at room temperature and eluted with 0.01 M ammonium bicarbonate in a growing and discontinuous gradient of NaCl (0.15 M, 0.25 M, and 1.5 M). The material was collected under a flux of 7 mL min−1 and monitored for absorbance at 280 nm. Peak I fraction (P-1) was then submitted to distilled water dialysis, lyophilization, and storage at −20°C for further testing.
Formulation of rhBMP-2 and P-1
Both proteins, P-1 and rhBMP-2 (Biozentrum, Germany), were diluted 1:1 with a neutral solution of phosphate tampon pH 7.4. Sufficient amount of monoolein gel to completely cover the critical bone defect was combined with 5 μg of each protein in a 7:3 (Monoolein:Water) proportion.
This study used 84 male Wistar rats (200–250 g body weight, 10–12 weeks) from Ribeirão Preto Central Vivarium at the University of São Paulo. The animals were kept under standard conditions and fed ad libitum under an experimental protocol approved by the Animal Care and Use Committee from the Medical School of Ribeirão Preto. These animals were housed in cages, containing five animals each, and allowed to acclimate for 1 week before surgery. The facility was maintained at 23°C ± 1°C with a light/dark cycle of 12/12 hr, initiating the light period at 7:00 AM.
Animals were divided into two large groups, with and without collagen treatment and each of these groups were divided into six treatment groups consisting of seven animals each. The treatment groups were: (I) 5 μg of pure rhBMP-2, (II) 5 μg of rhBMP-2/monoolein gel, (III) pure monoolein gel, (IV) 5 μg of pure P-1, V) 5 μg of P-1/monoolein gel, and (VI) critical bone defect controls.
All animals were anesthetized by an intramuscular injection of Xylazine (5 mg/kg) and Ketamine (75 mg/kg; Agibrands do Brasil LTDA-Campinas, São Paulo, Brazil). Gauze soaked in physiological saline solution (0.9%) was applied on both eyes during surgery to avoid corneal drying.
To expose the calvaria bone, a 1 cm sagittal incision was made in the calvaria central region using a scalpel blade number 15, previously mounted on a scalpel number 3.
A 6 mm wide critical bone defect was made in the left posterior region (parietal bone) of the exposed calvaria using a trephine bur adapted to a counter-angle head (Kavo, São Paulo, Brazil) with the aid of an electric implant motor (physiological saline solution).
Tissues were sutured after the surgical procedure with the animal skin and the periosteum repositioned to the original position. Sutures were made using silk thread 4.0 (Ethicon, Johnson & Johnson, São José dos Campos, SP, Brazil) and the margins of the flap were properly brought together. Sutures were not removed. Each animal then received an injection of 0.1 mL/100 g weight of small size veterinary pentabiotic (Fort Dodge®, Campinas, SP, Brazil) and an intramuscular injection of Banamine (Flunixin Megumine, 0.5 mg in 0.1 mL, NLS Animal Health) as an analgesic.
After 4 weeks from the creation of the critical bone defect, the animals were anesthetized with 37.5% urethane (using 1.5 g/kg body weight). The animals were first perfused through the left ventricle with 0.1 M phosphate buffered saline (PBS) at pH = 7.4. After about 200 mL of PBS was infused, this was followed by a 4% paraformaldehyde solution in PBS. Perfusion was assisted with an infusion pump (550 T2 Samtronic, Brazil).
Calvaria were removed after perfusion and the soft tissues were carefully removed from bone. These fragments were immersed in the perfusion fix solution for 24 hr and later decalcified using 0.5 M EDTA with a solution change every 2 days. After decalcification, which varied from 15 to 30 days, acid action was neutralized after 24 hr in a 5% sodium sulfate solution. The tissue blocks were dehydrated through gradual exposure to ethanol: 70% (overnight), 80, 85, 90, 95, and 100% (2 hr in each concentration). Bone blocks were immersed in equal parts of alcohol and xylol (overnight) and cleared in xylol, with three changes each for 2 hr. All blocks were later embedded in paraffin. Semi-serial cuts of 6 μm width were made in each sample and stained by Masson's trichrome and hematoxylin-eosin for later analysis in a light microscope.
The histological sections of the critical bone defect were observed using a grid containing 100 equidistant points in each analyzed field, adapted to the eyepiece of a light microscope (Leica DMRB, Germany) and connected to a digital camera (Olympus DP11, USA). The percentage of newly formed bone was measured using a differential point-counting method (Weibel et al., 1966). Ten sections per defect were analyzed.
A normal distribution was found for all data (Shapiro Wilk, P > 0.05). Factorial ANOVA test was made with two fixed variation factors (groups and use of collagen) and Tukey–Kramer test was made to compare each group (P < 0.05).
All results found in this study are presented in Table 1 and Graph 1 as means, standard deviations, and Tukey–Kramer's test results (P < 0.05). Group I (rhBMP-2) associated with collagen gelatin presented higher levels of newly formed bone (P < 0.05) than all other groups. Also, with collagen gelatin, Groups I and II presented significant higher levels of new bone formation (P < 0.05). When the collagen gelatin was not used, Group I presented higher levels of newly formed bone (P < 0.05).
Table 1. Means and standard deviation (SD) of bone trabeculae volume fraction (%) and Tukey–Kramer's test results of all six groups, with and without collagen application
Tukey-Kramer's test [Mean (SD)]
Critical value = 2.86313; P < 0.05. Within columns, different letters indicate statistical significance (Tukey–Kramer's test).
I (Pure rhBMP-2)
17.9 (6.8), A
26.0 (1.2), A
II (rhBMP-2/monoolein gel)
14.8 (7.1), B
21.5 (10.0), B
III (Pure monoolein gel)
7.6 (5.4), D
8.2 (5.7), E
IV (Pure P-1)
13.0 (5.8), BC
11.3 (7.7), DE
V (P-1/monoolein gel)
14.1 (6.6), B
16.5 (7.8), C
VI (critical bone defect)
10.2 (5.1), CD
14.0 (6.4), CD
Qualitative Analysis of Animals Belonging to 4-Week Group Without Collagen
After 4 weeks, animals with pure monoolein gel (Group III) application showed the presence of a small amount of newly formed bone in the periphery of the bone lesion. The central region of the critical bone defect presented a large amount of fibrous connective tissue and blood vessels, with few inflammatory cells (Fig. 1A). When pure P-1 was applied, a small amount of newly formed bone was found, especially in the central region of the lesion. Also, organized fibrous connective tissue and blood vessels were found in this region (Fig. 1B).
In Group V, with P-1 protein and monoolein gel as carrier, newly formed bone was found in a small amount in the periphery of the lesion. Organized fibrous connective tissue was found in the center of the lesion representing an extension of periosteum with blood vessels (Fig. 1C).
When pure rhBMP-2 was applied (Group I), newly formed bone was found with remodeled bone tissue in the lesion's periphery. The center of the lesion contained connective tissue as an extension of the periosteum and was characterized by numerous capillary and blood vessels, and in some animals the presence of multinucleated cells in connective tissue area (Fig. 1D). In the rhBMP-2 associated with monoolein gel Group II, newly formed and well trabeculated bone was found with the presence of osteocytes in ample osteocytic lacunae with an abundant number of osteoblastic cells around it. This newly formed bone was mainly found in the periphery of the bone lesion along with an organized as vascularized connective tissue (Fig. 1E).
It was found in the critical bone defect control Group VI, without the application of any material, the formation of thin bone trabeculae, permeated with osteocytes and involved in wide osteocytic lacunae. Different cells were also found in the peripheral layer, near the periosteum, mainly osteoblastic cells with reduced volume, indicating a modest activity of bone formation. Connective tissue was rich with blood vessels and fibroblastic cells. This tissue represents a continuance of the periosteum with small alterations (Fig. 1F).
Qualitative Analysis of Animals Belonging to 4-Week Group With Collagen
In the Group IV, where pure P-1 was used with collagen, it was found the formation of a thin layer of trabecular bone with osteocytes and wide osteocytic lacunae, involved by thick periosteum on one side and continuing throughout the lesion. Different cells were also found in periosteum layer, mainly reduced volume osteoblasts. Connective tissue was rich with blood vessels and fibroblastic cells. This tissue represented a continuance of periosteum, despite the presence of many alterations (Fig. 2A).
When rhBMP-2 was applied with collagen (Group I), extensive bone formation was found and surrounded by connective tissue with numerous medullary cavities and Haversian canals. Osteocytes were present in several lacunae indicating intense bone remodeling activity. Connective tissue can be characterized as thick and organized, rich in blood capillary, and vessels with different diameters (Fig. 2B).
Small amounts of newly formed bone were found when pure monoolein gel was applied with collagen (Group III). Also, some blood vessels were encountered with few osteocytic lacunae and with one side presenting developed periosteum. Connective tissue had few cells and blood vessels with various calibers (Fig. 2C).
When the association of P-1/monoolein gel (Group V) was used with collagen, trabecular bone was observed with wide osteocytes lacunae involved by periosteum and connective tissue contained fibroblasts, wide blood vessels, numerous capillary, and osteoblasts. (Fig. 2D).
Group 2, rhBMP-2/monoolein gel with collagen, had the presence of newly formed bone with lacunae containing osteocytes without Haversian canals and few cells. Fibrous periosteum with blood vessels of varying diameter was found. Connective tissue was thick and fibrous, with numerous cells, blood vessels, and capillary (Fig. 2E).
When the critical bone defect was made and collagen applied, the presence of osteocytes inside wide osteocytic lacunae was found, characterizing remodeling bone. Osteoblastic cells were also found in the periphery near the periosteum. Connective tissue was rich with blood vessels of varying diameters and fibroblastic cells (Fig. 2F).
The objective of the present study was to evaluate new bone formation in critical bone defects comparing two proteins, rhBMP-2 and P-1, the use of a carrier (monoolein gel) and/or a collagen gelatin coating. The bone healing process is associated with the presence of osteoregenerative cells (osteoblastic cells), osteopromotive proteins (growth factors, like BMP-2,4,6,7), and local environment (supplied by a material carrier) (Ripamonti, 2006). This study used rhBMP-2, previously described in literature (Issa et al., 2008; Schnettler et al., 2009; Wang et al., 2009) as a protein presenting osteoinductive capability, and a protein extracted from Hevea brasiliensis (P-1), which is currently being characterized. When the surgical area was covered with collagen, a higher amount of newly formed bone was found with the exception of Group IV with pure P-1. One possible explanation is that when covering the surgical area with collagen, the migration of soft tissue may be blocked, which is undesirable for bone repair (Thoma et al., 2009). Collagen is a biocompatible polymer and is present in large amounts in bone tissue and acts as a scaffold for new bone formation. It is also in vivo degraded and reabsorbed, allowing new bone formation where it was applied (Saito et al., 2005).
According to histomorphometric analysis, there was more bone formation in groups where rhBMP-2 was applied (I and II), especially when collagen gelatin was used to cover bone surface. The osteoinductive capability of rhBMP-2 leads undifferentiated mesenchymal cells to differentiate into osteoblasts and to synthesize bone (Riley et al., 1996). To optimize osteoinductive properties of rhBMP-2, substances able to maintain rhBMP-2 in the surgical area, such as the monoolein gel carrier (Issa et al., 2008), or inhibit undesirable cells from migrating into the bone defect area as collagen (Thoma et al., 2009), may be used. New bone formation occurred in the central area of the defect with P-1 protein and monoolein gel treatment. When the monoolein gel was used alone, the new bone was formed in the periphery of the defect.
This study used 5 μg of rhBMP-2 and waited 4 weeks until intracardial perfusion. Yasko et al. (1992) reported in their experimental study that a critical bone defect with 5 mm was regenerated after application of 11 μg rhBMP-2; however, 1.4 μg of rhBMP-2 was able to produce new bone tissue but not enough to close the bone defect. High dosages were used by Inoda et al. (2004) and showed that 50 μg rhBMP-2/collagen for a critical bone defect of 4 mm in rat skulls was able to promote new bone formation, but did not show advantages in relation to smaller and sustained release dosages as shown by Wang et al., 1990, who tested rhBMP-2 dosages from 0.5 to 115 μg. Saito et al. (2005) reported that 3 weeks after rhBMP-2 application (10 μg) associated with an injectable polymeric delivery system (PLA-DX-PEG), rhBMP-2 retention was found on the femur surface.
In the present study, to evaluate newly formed bone, a 6 mm diameter critical bone defect was performed with trephine bur under abundant saline solution irrigation. Nowadays, current literature has presented different carriers and scaffolds for BMP delivery with the aim to obtain good osteoprogenitor colonization and consequently induce bone repair. Using an in vivo rat calvarial model, many studies are found in literature involving different doses of BMP associated with different carriers, such as collagen (Hollinger et al., 1998), gelatin (Hong et al., 1998), poly (lactic-co-glycolic acid) (Cowan et al., 2007), fibrin (Chung et al., 2007), ß-tricalcium phosphate (Ruhe et al., 2004), and hyaluronic acid (Kim et al., 2007). In all these studies, BMPs were able to promote the bone healing process and the carriers improved the processes.
Aiming to study monoolein gel as a carrier, more new bone formation was found when P-1 was associated with a carrier (Group V) when compared to using the protein alone (Group IV) and this difference was significantly different when the collagen gelatin was used (P < 0.05, Table 1). This condition was not obtained when using rhBMP-2 protein and the association between rhBMP-2 + monoolein was less inductive of bone formation than rhBMP-2 alone (P < 0.05, Table 1). The use of monoolein gel alone (Group III) induced the least amount of bone formation among the groups analyzed in this study and this difference was significant when the use of a collagen gelatin was associated (P < 0.05).
When compared with control Group VI, all groups using rhBMP-2 (Groups I and II, with and without use of collagen) presented significantly more bone formation (P < 0.05). On the other hand, the use of P-1 protein was not significantly more effective for new bone formation when compared with the control group (P > 0.05), except when it was associated with monoolein gel and without using the collagen gelatin (P < 0.05).
Within the limits of this study and using 6 mm critical bone defect in rat calvaria as an experimental model, it can be concluded that rhBMP-2 allowed more new bone formation than P-1 protein and this process was more expressive when the bone defect was covered with collagen.