Production And Appliance Of Compound RhBMP-2/nHA/Co Material

Periodontitis is one of the most common diseases in oral medicine, and therepair of periodontal defect is targeted to periodontal membrane, cementum andalveolar ridge. Tissue engineering, which three main elements are seeding cells,degradable vector material and regulating factors, provides new method oftreatment for periodontal defect regeneration.Generally, the tissue engineering of periodontics also includes the threemain methods of Growth factor-Vector, Seeding cell-Vector and Seedingcell-growth factor-vector. There are many clinical problems to be solved for thelater two kinds (the origin of seeding cells and its immunology etc.). So the keyresearch field is to accelerate the periodontal regeneration with appropriate vectorcontaining foreign growth factors.The ideal vector materials should have the following characters: (1) goodtissue compactibility;(2) bio-degradebiligy and the control for theirdegradation;(3)good bone conductibility and bone inductivity;(4) without antigen;(5) The surface should be in favor of maintaining the cell morphology and mould,and help the cell in adherence and proliferation;(6) the vector should havethree-dimensional, multi-hole structure, and the size of holes should be in favor ofthe transportation of nutrients and metabolic products;(7) the vector should has itsintensity;(8) the vector should take growth factors and other active materials;(9)the vector should be easy to be sterilized.BMP has no specialty in species, and is a special local growth factor. Itmainly induces and stimulates the differentiation and proliferation ofmesenchymal cells to form osteoblast line. BMP can induce the stem cells in PDLto differentiated into cementoblast and osteoblast. BMP-2 is the strongest factor toinduce bone formation, but the purified BMP would be hydrolyzed implanted intothe body sooner, so that its ability of inducing is weakened. So BMP is oftencompounded with vector materials to reduce the rate of hydrolyzation.Hydroxyapatite(HA)is the main abio-element in marrow and teeth ofanimals. The collagen fibers is regularly arranged around hydroxyapatite, and thealveolar ridge be up to 97% of hydroxyapatite. HA ceramics has very goodbio-compatibility and bone conductibitlity, be widely used in bone regeneration.The conductibitlity of HA can provide good environment for cell growth, while itsmulti-hole structure can be benefit for cell growth, tissue regeneration andvascularization, which is fit for the require of bone tissue engineering. Nano-HAceramics is similar with natural bone, can improve the mechanical character ofbone implants and be used as the vector of BMP.Collagen is a kind of fiber protein. Collagen Ⅰhas antitension and can befound in all parts of the body. Collagen can not be only formed the framework ofextra-cellular matrix, but can interact with cells to influence the morphology,proliferation and differentiation of cells. Collagen can be used as the effectivevector of BMP, and its degradability makes it an important material in tissueengineering.Objectives of this research: To composite rhBMP-2, nHA and collagentogether, and evaluate the bio-characteristics of this compound material. In thisway, we want to use the advantage of every one to make it a new substitute ofbone regeneration and vector material.Experiment I Preparation of CompoundBio-MembraneExtraction of Collagen Ⅰ: Adult cattle Achilles's tendon was first frozened,and then cut into 0.1mm slice. It was put into 0.05 moL· L-1 acetic acid(pH 3.2),and digested with pepsin(100mg·g-1), and then filtrated and centrifuged. The upperlayer was salt outed with NaCl, and centrifuged after the separation of whitedeposition substance. The deposited collagen was dissolved in acetic acid, anddialysed with 0.01 moL· L-1 pH7.4PBS for 96h, then dialysed with distilled wateruntil there was no Na+,Cl-in the solution. The dialysed collagen was dehydratedwith PEG and concentrated as liquid collagen, which was then coupleted withglutaraldehyde.Synthesize of nHA: The nHA was synthesized with precipitation. TheCa(OH)2 and H3PO4 was mixed together in the ratio of Ca/P as 1.67, and theH3PO4 solution was put into Ca(OH)2 glycol solution under mixed. After 2h'smixed, they were filtrated, drying and then calcined for 2h under 300℃,600℃,800℃ respectively.Preparation of nHA/Co Membrane: 0.5g nHA powder was surged withultrasonic for 10min, and dissolved in 75ml,0.1mol· L-1 HCl. Then, 225mlcollagen type I was added into the former solution and whisked and diluted withdistilled water to 800ml, then 0.025mol· L-1 NaOH (PH=12.5)was put into ituntil the pH was about 6.2, and then remained at 7.2 for 10min. The uppersolution was wiped off by eccentricity, and the sample was frozen dried for allkinds of test.Results: 1) SDS—PAGE showed that the prepared collagen had onestrip(about 98kD), saying that it was collagen type I. SEM showed threedimensional reticulational structures of the collagen membrane;2) The size ofhole of the synthesized nHA was 10nm×50nm～20nm×80nm, resembling withthat of natural bone;3) The surface hole and size of compound material wassymmetrical, and nHA granule was equally distributed to the matrix of collagenand combined with collagen finely, with the mean size of granule 60nm;4) Thechemical composition of compound material was similar with that of natural bone,mainly by collagen and carbonic nHA.Experiment II Biocompatibility of CompoundMembraneMethods:1) Extraction and culture of BMSCsFour-week old rabbits, weighing 2.5～3.5kg, was extracted 3mL marrow onboth femurs, then they were cultured under DMEM medium containing 15% FBSand made into single-cell suspending solution. The cell was inoculated into 50mLculture bottle(3×106) and cultured under 37℃,5%CO2. The medium was changedhalf 5 days later and changed completely every 2～3days. The third generationwas digested with tripsin and innoculated into 12 hole orifice (5×104)under themedium containing DEX, Vitamine C and B-sodium glycerophosphate for 21 days.When the cell growed colonily, the Von Kossa staining was performed.2) Co-culture of Osteoblast and nHA/Co:The nHA/Co membrane was sterilized with Co60 and implanted the BMSCswith 5×106·mL-1 for culture. The medium was changed every 2～3days for 5executive days. Then the cell was washed with PBS and fixed in 4% glutaral andHE staining. The sample of cell for SEM was prepared for examination ofadherence and morphology.Results: 1) The cells showed positive staining of Von Kossa after 21days ofinduction, proving that it had the ability of osteogenic. 2)The co-culture: Lightmicroscope showed that cells adhered on the surface and margin of membrane andlinked to form a slice;SEM showed that many BMSCs adhered on the margin ofmembrane and there was some matrices in some area. So the membrane had goodbio-compatibility and no toxic to the cells.Experiment III Bone Regeneration Induced byMembraneMethods: Sixty male four-week athymic mouse, weighing 17～19g, wererandomly divided into nHA/Co group and rhBMP-2/nHA/Co group. Themuscular sac of both thigh was implanted with two materials. And ten mice weresacrificed respectively 10,20,30days after the surgery. Then the thigh was takensoft X ray and made into routine slice for HE staining.Results: There was no infection and rejection. nHA/Co group showedimplanted gelosis on the skin surface and disappeared 10days later;rhBMP-2/nHA/Co group showed the same gelosis and gradually increased with time, untilthe hardness like bone 20days later.Soft X ray showed that nHA/Co group had no high density area in themuscle while the rhBMP-2/nHA/Co group had high density area in the muscleand calcification spot. Focal high density area could be seen 20days later andreached the highest 30days later, which showed bone-like structure and bonetrabicula to form reticulation.Tissue Observation : 1)nHA/Co group showed degradation and resorptionof membrane 10days after the surgery and some infiltration cells, with a lot ofneutrophil cells and lymphocyte infiltration, and also some multi-nuclimacrophages and foreign-body granuloma.2) Focal cartilage cells formed 20daysafter the surgery and calcification and new bone formation. Most of the membranewas degraded.3) A lot of woven bone and lamellar bone formed 30days after thesurgery, and combined into new bone trabecula and marrow, while most of themembrane was resorpted at the time.Experiment IV Experimental Repair of MandibleDefect with Compound MembraneMethods: 20 healthy adult New Zealand rabbits were prepared rectanglebone defect of about 1.0cm×0.5cm at middle part of bottom edge of bothmandibles. The right side was the experimental one, whose defect was coveredwith nHA/Co compound membrane;while the left group was sutured without anymembrane. The animals were sacrificed 28d, 56d after surgery respectively, theboth mandibles were taken soft X ray;and routine HE staining for lightmicroscope examination.Results:Naked eyes observed healthy rabbits with the incision healing at first stage.Soft X ray showed little volume of new bone in the control group, and therewas still the nick 56 days after the surgery. While, there was bone-like density inthe bone defect area 28 days after the surgery, and the density increased untilcompletely healed 56 days after surgery. The new bone combined with the oldbone with a borderline.Tissue Observation:Control Group: there was much fiber in the defect area 28 days after thesurgery, and also some multi-nucli macrophages. There was some new bonetrabecula and osteoblast around the bone trabecula;The density of new bonetrabecula increased 56 days after the surgery and there was even some layer bone.nHA/Co Group: The membrane were still there 28 days after the surgery,and the new bone combined with each other to form reticulation structure. Themembrane had been resorpted 56 days after the surgery, and the defect area hadbeen substituted by new formed bone.Experiment V Periodontal Regeneration byCompound MembraneMethods: 8 adult male dogs were selected in this experiment.rhBMP-2/ nHA/Co membrane was sterilized by Co60 before the surgery. A "U"like bone defect about 4mm was prepared on the buccal side of molar area until5mm under the enamel-cemtum junction. The exposed periodontal membrane andcementum was scraped and planned. The compound membrane was put on rightside and the left side was empty as control side. Four animals were sacrificed 4, 8weeks after the surgery respectively. Sixteen row CT ray was taken showed thenew bone formation, and routing HE staining was made to observed undermicroscope.Results:The animals grow healthy, with the gingival healing well and PD<1mm.CT showed new alveolar bone formation and the ridge growed to its originalheight. While , there was not new bone formation in the control group.Tissue observation showed granuloma formation at the site of implantationfour weeks after the surgery, and the membrane was degraded. There was muchneutrol cells, lymphocytes and multi-nucli macrophages, with much fiber aroundthem and without any new cartilage. 8 weeks after surgery, most of the membranewas absorbed and the cell infiltration decreased. The fiber began to grow andsome bone-like matrix and cartilage began to appear. The junctional epitheliumgrow fast to form long junctional epithelium.Experimental Group: There was many neutrol cells and lymphocytesinfiltration, and the fiber grow fast with some cartilage-like matrix and cartilage.There was some calcium precipitation and multi-nucli macrophages as well. Thenumber of infiltrated cells decreased 8 weeks after surgery, and there was muchfocal cartilage cells and cartilage area, with new bone trabecula combined withform reticulational structure. The new bone was linked with alveolar bone with aborderline between them, and some periodontal fiber and cementum.The upper experiments showed that:1. The compound HA granule was similar with that of natural bone, and fit therequire of nano-size for HA in natural bone;The extracted collagen is highlypurified with low antigenicity;The HA could combined with collagen to formtightness bonding.2. nHA/Co has good bio-compatibility, without any cell toxicity and can be usedas the bone substitute material.3. Collagen, nanoHA are both good vectors of BMP. rhBMP-2/nHA/Cocompound membrane has precise volume of rhBMP-2, with no effect on itsbone inducement activity.4. The compound nHA/Co membrane has a good ability of bone induction andbone formation.5. rhBMP-2/nHA/Co could be completely degraded 6-8 weeks after implantationand accelerate the periodontal regeneration.Conclusions: rhBMP-2/nHA/Co compound membrane can be used as a newbio-active material and vector material as bone substitution in tissue engineering.