Effect Of Surface Microtopography On Bone-formation In The Interface Of Bone And Implant

It has been a long time for human to explore dental implant. Until 1982, firstly, the concept of osseointegration was held on the North-American Conference in Toronto, many researchers paid close attention to dental implant as an authentic functional prosthesis. Due to its fine restorative outcome that traditional denture unequal to and its esthetic effect, more and more clinic dentists have applied implant denture to restore these cases that miss teeth and defects that traditional denture hard to repair. Striking success were obtained after several decades of research and application. However, scholars and experts have been striving to unravel how we can maintain long-term stability and function after inserting dental implants into alveolar bone and understand its correlated mechanism and prerequisite. By clinic observation , we understand many influential factors exist in long-term stability of dental implant , mainly including ① physio-chemical property and biocompatibility of implantmaterial ,② design of implant configuration , ③ implant surface microtopography , ④host-related condition , ⑤surgical procedure ,⑥design of superstructure , ⑦loading ,⑧time et al ,the key of long-term success of dental implant is early and extensive bone-formation with peri-implant bone .In previous animals experiment and clinic observation, scholars have found that surface microtopography of implant influences distinctly the rate of bone-formation and the ratio of bone-implant contact .Is it because uneven surface affording to different bony area or because physical configuration playing different role in inducing bone-information ? Recently,several foreign studies reported implant surface microtopography influences proliferation , differentiation of osteoblasts , however, the mechanism has been still unclear . Furthermore , we understand that bone-formation of peri-implant causes mainly by mineralization of extracellular matrix secreted by osteoblasts . Nevertheless , cytokine plays a critical role in regulation of bony formation , growth and repair . Presently , some domestic and foreign scholars have survey cytokines such as BMP(bone morphogenic protein , TGF(transforming growth factor) , IGF(insulin-like growth factor) , FGF(fibroblast growth factor) and PDGF(platelet derived growth factor) have important effecs of inducing bone-formation at cellular level . In preivous reports , only was shown that single cytokine or both cytokines effects to osteoblast, it was seldom reported that synergism of surface microtopography and cytokine to osteoblast . The purpoes of our study is to investigate influences of surface microtopography to bone-formation of osteoblasts and synergism of proliferation , differentiation of osteoblast with two factors while adding cytokine , to study further themechanism and regulating factors of bone-formation of peri-implant by surveying synergism of bone-formation of osteoblast with two factors , to provide theoretic and experimental data for clinic use .The paper contains three parts , part Ⅰ : the study of bone formation interface of two different surface microtopography titanium implant in vivo .part Ⅱ : influence of proliferation , differentiation of osteoblasts on different titanium surface microtopography in vitro . partⅢ: investigation synergism of proliferation , differentiation of osteoblast with two factors (TGF- β1 and surface microtopography).Part Ⅰ Objective: The aim of this animal study was to compare the effect of surface topography of two type of implants on the response of the bone formation at the bone-implant interface.Methods: Three male adult rhesus monkeys (Macaca mulatta) were used in this experiment to establish the mandibular edentulous model. The two second premolars and the two first molars of each monkey were all extracted and the implants were inserted after 3 months. Six two-part solid screw CDIC implants ( 10mm in length, 4mm in diameter, provided by China Dental Implantlogy Center ),and six two-part solid screw ITI-TPS implants (10mm in length, 4.1mm in diameter, provided by the Straumann Group of companies ) were used in this experiment. The two type of implants were different in surface topography. The surface of CDIC implants were machined (smooth surface), while the surface of ITI-TPS implants were titanium plasma sprayed (rough surface). On one side of the mandible of each monkey, a CDIC implant was inserted at edentulous area. The other side was inserted in an ITI-TPS implant. All the operations werecarried out by one operator.Results: All implants were stable after implantation. State of bone formation at bone- implant interface was investigated after 1, 2, 3 and 12 months. Clinical observation included state of gingival heath around implants, depth of pocket aroud the implants and movement level of the implants. X-ray and hisological studies were practiced to investigate bone formation at bone-implant interface after 1, 2, 3 and 12 months. The results showed that titanium implants with TPS coating had an average bone-implant contact (BIC)significantly higher than CDIC implants in the early healing phase. Both the two type of implants can reach a satisfactory BIC as time goes on.Conclusion: This experimental finding have led to the conclusion that rough surface of ITI-TPS implant is better at accelerating apposition of bone than smooth surface of CDIC implant in the early healing phase.Part Ⅱ: Influence of different titanium surface microtopography on proliferation , differentiation of osteoblasts in vitro .Experiment Ⅰ: Establishing osteoblastic lineage in vitro Purpose:In order to study the Influence of proliferation .. differentiation of osteoblasts on different titanium surface microtopography in vitro,we establish osteoblastic lineage in vitro from murine. SD feta 1 rat calvaria were isolated and cultured by the method of tissue-fragment migratig growth, fibroblast were used as a control. Result: Through invert microscope , continual morphologic observation shows that the osteoblasts are spindle, triangle or polygon, increasing cell body , rich cytoplasm, oval nucleai characteristic secretory granule , colonyshape occur with growth.However, fibroblast occurs long- spindle , whirlpool shape .The third passage osteoblasts were examined alkaline phosphatase-specific activity(ALP),and production of osteocalcin(OC).The contents of ALP and OC of test group were high significantly than those of control group .It indicated that cells which were isolated and cultured are osteoblasts.Experiment Ⅱ: Influence of proliferation , differentiation of osteoblasts on different titanium surface microtopography in vitroPurpose: In order to study the Influence of proliferation , differentiation of osteoblasts on different titanium surface microtopography in vitro, commercially pure titanium discs were fabricated to be 15mm in diameter , 1mm thick with three different surface roughness: a relatively smooth , machined surface(G);a sandblasted surface (SB);and a titanium plasma-sprayed(TPS). Roughness value(Ra): G surface 0.48 um, SB surface 2.8um, TPS surface 5.35um. The third-fifth passage osteoblasts was poured onto all 3 groups discs in 12-well plate, plastic surface were used as a control.Result:①Cell morphology : Cell morphology varied with different surface roughness were examined by SEM. Osteoblasts adhered tightly to three titanium surface,spreading in all directions. With G surface, osteoblasts were spindle-shaped with little cytoplasm. Osteoblasts were cubic, multangular and abundant cytoplasm on SB surface and TPS surface.②Cell proliferation assay: Cell proliferation on the different titanium surface were measured by the MTT Assay. The number of cells increased froml-7days on all discs, , while cell proliferation was significant higher on on SB and TPS surface than G surface (P<0.05) and it was not statistically significant difference between SB and TPSsurface ( P>0.05 ) . ③ ALP measurements: An increase in alkaline phosphatase activity was detected on SB and TPS surface than G surface(P<0.05) .④Osteocalcin production: Synthesis of extracellular matrix proteins was more abundant on SB and TPS surface than G surface (P<0.05) .Part Ⅲ :reaearch of synergism of proliferation , differentiation of osteoblast with TGF- β1 and surface microtopographyPurpose:To investigate synergism of proliferation , differentiation of osteoblast with TGF- β1 and surface microtopography.Adding TGF- β1(10ng/ml) to all 3 groups discs in 12-well plate in presence of osteoblasts on 1,3,5,7,10 days, plastic surface were used as a control.Result:①Cell proliferation assay: Cell proliferation with TGF- β1 and different titanium surface were measured by the MTT Assay. Cell number was no change adding TGF- β1 compared with unadding cell factor froml-5days on G discs, while cell proliferation was significant inhibited from 7days(P<0.05) .After 24 hours of adding TGF- β1 ; cell proliferation was no change ,while cell proliferation was significant inhibition on on SB surface than unadding TGF-β 1 (P<0.05) . Cell number was not statistically significant difference adding TGF- β1 compared with unadding cell factor from1-5days on TPS discs, while cell proliferation was significant inhibited from 7days(P<0.05). (3)ALP measurements: An increase in alkaline phosphatase activity was detected on all four discs from 24 hours(P<0.05),while alkaline phosphatase activity was not statistically significant difference on all four discs from3-5days.Adding TGF- β1 ,an increase in alkaline phosphatase activity was detected on SB and TPS surface than G surface (P<0.05) .④Osteocalcin production: On G surface,an increase in osteocalcin production was detected after adding TGF- β1 (P<0.05) from l-3days;while osteocalcin-secrtion has increasing tendency on SB and TPS surface after adding TGF-β1,up to lOdays, the tendency was significant (P<0.05) .After adding TGF- β1, osteocalcin production was more abundant on SB and TPS surface than G surface and control (P<0.05) .