Bone replacement therapy is commonly used in an oral surgeons office to reconstruct the jaw bone due to bone loss from disease, or to support the existing jawbone when performing tooth implant procedures (Zeng 1991). Coral scaffolding is an emerging form of bone graft scaffolding on which surgeons have been able to regrow human bone with promising results (Bensaid 2003). This recent advancement solves problems with bone donation rejection, and scarcity of donation material. Research into this bone replacement material began in 1974 with animal trials, and moved to human trials in 1979. Results have shown promising morphological similarities in resulting bone porosity and structure due to the composition of the implanted coral scaffolding. Important osteogenic abilities of the regrown bone have been confirmed via osteoblast differentiation. However sufficient long term effectiveness of this coral scaffolding on osteoclastogenesis have not been studied. The ability for bone to produce working osteoclasts is necessary due to their role in bone resorption, or the breakdown of bone. A series of experiments has been proposed to assess the ability of HMSC derived osteoblasts to activate their RANK Ligand receptors to initiate osteoclastogenesis. Gene sequencing and morphological studies will be used to insure osteoblast presence. RT-PCR and immunohistochemistry will be used to assess mRNA and protein expression of RANKL, RANK and OPG.
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Bone resorption is a very important function of normal, healthy bone. When bone cells die from disease, trauma, or a bone fracture, bone regeneration must take place to replace the lost bone. This process is known as bone remodeling. Bone remodeling, also known as bone metabolism, is a constant cycle that occurs on trabecular and certain cortical bone. It destroys and creates bone using a set of specialized cells. Ossification (bone creation) is controlled by osteoblasts (Bone 2010). Osteoblasts are derived from differentiated osteogenic cells of mesenchymal origin in the periosteum. Osteoblasts can be characterized by the expression of RANKL and alkaline phosphatase. Bone regeneration however cannot occur directly following an injury because there is simply no room for osteoblasts to insert themselves to begin the rebuilding process. First, the old dead bone tissue must be destroyed and cleaned out to make room for the osteoblasts. This process of bone destruction is called resorption, and is carried out by a very specialized cell, the osteoclast. Osteoclasts are large, multi-nucleated cells (Tolar 2004) that are found in small depressions along the surface of the bone called Howship lacunae. When osteoclasts accumulate in one area their enzymes begin to erode the bone, creating these small depressions. Bone resorption also plays a very important role in the human body's homeostasis. As the macrophage cells devour the dead bone cells, it releases calcium into the blood supply to help meet the body's metabolic needs. This give and take relationship between osteoclasts and osteoblasts allows bone to alter its size and shape until it finally reaches its full adult form.
Osteoblasts are naturally found on the outside of the bone where they create a sheet of cells covering the surface of the bone. It is from this film of cells that bone remodeling is controlled by the signals produced by the osteoblasts. The primary signal that osteoclasts produce towards the bone surface is RANKL which promotes osteoclastogenesis. This transformation is ultimately controlled by vitamin D levels in the bloodstream. Vitamin D produced by the skin from sun exposure travels through the bloodstream to the kidneys where it is then converted into its active form 1,25-dihydroxy vitamin D by the parathyroid hormone. 1, 25-dihydroxy vitamin D balances serum calcium levels to proper levels via resorption of calcium rich bone (Holick 2005). Activated vitD interacts with it vitamin D receptor in osteoblasts which produces the receptor activator of RANKL (Figure 1).
RANKL stands for receptor activator of nuclear factor kappa-B ligand, and is also known as ODF, the osteoclast differentiation factor. RANK Ligand is part of the cytokine family which operates as one of the two factors in osteoclast differentiation and activation. It is a protein encoded in humans by the TNFSF11 gene and is found on the outside of osteoblast cells. When RANKL is produced by osteoblasts it initiates the phosphatidylinositol-3 kinase (PI3K)-Akt pathway causing the monocytic precursor cells from the bloodstream to differentiate into adult osteoclasts. (Figure 2, 2004)
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Monocytic precursor cells are introduced to the area via blood vessels formed through the bone matrix. They interact with osteoblasts and begin differentiation into osteoclasts by producing two proteins on their cell membranes, TNFR1 and RANK. RANK matches with its cognate receptor RANKL on the outside of the osteoblast cells and the cell becomes committed into an osteoclastic lineage (Atkins 2003). Activation of the RANK/RANKL interaction can be prohibited by osteoprotogerin (OPG) binding to RANKL. OPG is also referred to as the osteoclastogenesis inhibitory factor (OCIF) and is coded for by the TNFRSF11B gene (Hofbauer 2004). The adaptor molecule TRAF6/TGF-activated kinase (TAK1) is recruited in the cell to activate several pathways leading to differentiation. Two of these pathways, the nuclear factor-B (NF-B) and the N-terminal kinase (JNK) signaling pathway, are indispensable in order for monocytic precursors to differentiate into osteoclasts. Activation of these two pathways produce several osteoclastogenic transcription factors including NFxB and FRa1 [(Figure 2 (lower) 2004] Proper production of RANKL is essential in osteoclast differentiation induction, and must be regulated in order to sustain bone homeostasis. If bone resorption rates were to exceed the ability of osteoblasts to build new bone, osteoporosis can occur. If the tables shift in the opposite direction, where more bone is produced than destroyed, the resulting condition is osteopetrosis (Cell 2004). The majority of bone diseases found in humans are somehow related to the regulation of osceoclast/osteoblast populations (Suzuki 2008).
Bone replacement therapy has a number of surgical applications from osteoporosis treatments to emergency bone loss procedures. The most common use is in tooth implantation preparation. Often, when a patient seeks to have a replacement tooth implanted into their jaw, there is not sufficient bone to support the implant. A surgeon will then have to use a sort of bone graft in order to increase the amount of bone in the area. After the bone heals the oral surgeon will then drill a hole for the implant to rest, and the implant is inserted. Drilling is a very traumatic procedure causing increased blood flow in the area. In order for the bone to heal correctly around the implant, the dead bone must be resorbed before ossification can occur. Current methods of bone repair include transplantation of healthy bone from another donor, a healthy part of the patient's body, using a man-made composite, or a xenograft. These grafts come in the form of a bottle of granules that are mixed with a fibrin gel and pasted into the injured area. The fibrin gel houses the HMSCs and the wound is closed over. These implants take weeks, or even months to heal, so stabilizing the operation site with metal bolts, screws and plates is often necessary to stop the patient from reinjuring themselves while healing in more severe bone-loss patients. Human mesenchymal stem cells (HMSCs) are pluripotential stem cells that can be expanded in vitro without losing their bone cell making abilities. The stem cells are then allowed to proliferate into bone to replace missing regions around the scaffolding.
Each current method has its downfall. Donations from another patient risks rejection, and surgeons can only take limited portions of bone from the same patient. Research shows an alternative method, marine coral scaffolding (Bensaid 2003). Research into whether or not coral grafts could be used as suitable bone grafts in the early 1970s in animals and in 1979 in humans (Guilleman 1987). In 1991, researchers at the Sun Yat-sen University of medical Sciences used coral implants in clinical human trials with 22 patients. Using X-ray, histological and clinical examination, the coral proved to have a good tolerance and did not inhibit any sort of a rejection response from the host. The coral Porites lutea is naturally housed in its own calcium based porous infrastructure much like the makeup of spongy bone tissue. The structural and mineral composition of natural coral is very similar to human bone, but it cannot be implanted in its natural form. Once the coral pieces have been collected and sanitized, they are chemically treated under high temperature and pressure to convert their natural calcium carbonate matrix into useable hydroxyamatite (Ripamonti 1992). Normal human bone is comprised of up to fifty percent of a modified hydroxyl apatite mineral (Junqueira 2003)
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The rate of coral resorption relies heavily on the pore size and the interconnectivity of the implanted coral (Kuhne 1994). Once the coral hardens in the body into a new exoskeletal matrix, it forms pores ranging from 150 to 200 microns in diameter, the same pore size found in human bone (Demers 2002). This makes coral an ideal background for HMSCs to proliferate into due to its morphological and chemical composition. Once implanted, the natural bone's layer of osteoblasts begin to encroach on the wound site, dissolving the coral scaffolding as they multiply and replace the coral leaving no precursors of the non-human tissue behind.
While these differentiated cells may form a sponge-like morphology when proliferating, how effective are these newly formed bone cells at producing osteoclasts? Osteoclasts are characterized by a cytoplasm with a homogeneous, "foamy" appearance. This appearance is due to a high concentration of vesicles and vacuoles. These vacuoles are lysosomes filled with acid phosphatase. It is this acid phosphatase that is responsible for the degradation of the normal bone during bone remodeling.
When osteoclasts resorb bone they must degrade both the biotic and non-biotic components of the cell. When implanted on a coral scaffolding, there are no biotic components to destroy. This causes bone to grow quicker into coral scaffolding than in human bone. Though this may seem a positive characteristic, could this have more detrimental consequences when studied long term? Could this increased rate of absorption have somehow altered the osteoblasts ability to regulate osteoclastogenesis?
In August of 2010 a team of researchers at the Pham Ngoc Thach University of Medicine in Vietnam were able to culture and differentiate osteoblasts on coral scaffolding using human bone marrow mesenchymal stem cells (Tran et al 2010). MSCs derived from human bone marrow were transferred from culture onto a corral scaffold. They were then induced into osteoblasts with two growth factors, FGF9 and vitamin D2, in an osteogenic medium. However no research has yet been conducted on whether the use of the corral scaffolding effects the osteoblasts ability to induce osteoclastogenesis. In this study we will assess regrown osteoblasts' ability to induce osteoclastogenesis. First we will test the osteoblast' gene for possession of the TNFSF11 gene. Once this is confirmed, RT-PCR and ELISA will be used to detect RANKL and OPG mRNA and protein expression in cultured human osteoblasts on coral scaffolding compared to human bone. Immunohistochemistry will then be used to insure the produced RANKL proteins are embedded on the outside of osteoblast cells. When monocytic precursor cells are introduced to these osteoblasts, osteoclast differentiation should occur. Another set of experiments using the same procedures should be attempted growing the osteoblasts in 1, 25-dihydroxy vitamin D rich media to assess the effectiveness of higher concentrations of vitD on bone desorption rates on coral scaffolding.
Rat Mesynchmal Stem cells will first be isolated from bone marrow aspirates (Wlodarski 1990). Bone marrow will be collected by flushing the femur with 10 ml of a proliferation medium into a T75 culture flask. 24 hours later, the cells will be washed with PBS and fresh medium will be added. The medium will then be replaces every three to four days until the cell cultures reach 80-90% MSCs. The cells will again be washed with PBS and incubated and expanded at 37 C until the cells become detached (Liu 2009). The MSCs will then be forced to differentiate into osteoblasts with the addition of an osteogenic supplement and 10% fetal bovine serum in minimum essential medium. Von Kossa staining will then be used to view and compare the morphologies of these osteoblast cells. DNA will be extracted from the osteoblasts and prepared for sequencing. The gene section containing the TNFSF11 gene will be amplified, and a PCR will be performed. A PCR cleanup will then be performed to remove all primers before being sent for sequencing. Results will be used for a BLAST comparison to the known human genome, to ensure the presence of the TNFSF11 gene.
The osteoblastic cells will then be seeded onto 3 coral discs. Another 3 coral discs will be seeded with osteoblasts and will be treated with varying daily aliquots of 1,25-dihydroxy vitamin D, at 5% 10% and 20% concentrations (Cellular 2005). A final 3 plates of rat bone tissue will be seeded with osteoblasts. All of the experiments will be repeated 3 times. An alkaline phosphatase (ALP) assay will be performed every 3 days for 21 days to assess the amount of cellular activity in the growing bone cells. Microscopic studies of the cell/coral borders should show a proliferation of osteoblast cells covering the porous scaffolding. There should be a trend showing more ALP production and cell growth in the coral vitD treated discs than the non-treated, followed by the human bone control discs.
mRNA Expression in Induced Osteoblasts:
While the TNFSF11 gene should be proven present in the osteoblasts, mRNA analysis must be used to confirm that the osteoblasts can activate this gene upon increased levels of vitD. After proliferating on the various discs, total RNA must be extracted from the cell. Using specific gene primers for TNFSF11 will then be used for real-time PCR. If the cells are actively expressing the TNFSF11 gene there should be a positive result from the RT-PCR images. There should be more of the gene expressed in the vitD treated samples then in the nontreated samples.
Expression of several important osteoblast markers will be assessed using an immunoperoxidase technique with diaminobensidine as the active chromogen. Immunohistochemistry was performed using an immunoperoxidase technique with diaminobenzidine (DAB) as the chromogen. The osteoblasts will be relocated from the discs and incubated in a cytochemical medium consisting of 15 mg DAB and 0.01% H202 at 37'C for 75 minutes. Creating serial sections of treated cells will allow for RANKL, RANK and OPG examination.
Protein expression of RANKL, RANK and OPG:
Immunocytochemistry will be used to assess the protein expression in these osteoblasts. Rabbit polyclonal anti-human soluble RANKL antibody (PeproTech Inc., Rocky Hill, NJ, USA), rabbit polyclonal anti-human RANK antibody (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA) and mouse monoclonal anti-human OPG antibody (IMGENEX, San Diego, CA, USA) will be used to determine the expression of RANKL, RANK and OPG in these induced osteoblasts respectively.