M Nevins, DDS; DM Kim, DDS, DMSc; SH Jun, DDS, MS; Kevin Guze, DMD; Peter Schupbach, PhD; ML Nevins, DMD, MMSc

Abstract:
Previous research has demonstrated the effectiveness of laser-ablated microgrooves placed within implant collars in supporting direct connective tissue attachments to altered implant surfaces. Such a direct connective tissue attachment serves as a physiologic barrier to the apical migration of the junctional epithelium (JE) and prevents crestal bone resorption. The current prospective preclinical trial sought to evaluate bone and soft tissue healing patterns when laser-ablated microgrooves are placed on the abutment. A canine model was selected for comparison to previous investigations that examined the negative bone and soft tissue sequelae of the implant-abutment microgap. The results demonstrate significant improvement in peri-implant hard and soft tissue healing compared to traditional machined abutment surfaces.

Materials and Methods:
The current study was designed to examine the effects of two different implant and abutment surfaces on epithelial and connective tissue attachment, as well as peri-implant bone levels. Six foxhounds were selected for this study. Each dog received 6 implants in the bilateral mandibular premolar and first molar extraction sites, for a total of 36 implants. The sites were randomly assigned to receive tapered internal implants (BioHorizons) with either resorbable blast texturing (RBT) or RBT with a 0.3mm machined collar. In addition, either machined-surface or Laser-Lok microchannel healing abutments were assigned randomly to each implant. The abutments were placed at the time of surgery.


Results:
The presence of the 0.7 mm laser ablated microchanneled zone consistently enabled intense fibroblastic activity to occur on the abutment-grooved surface, resulting in a dense interlacing complex of connective tissue fibers oriented perpendicular to the abutment surface that served as a physiologic barrier to apical JE migration. As a consequence of inhibiting JE apical migration, crestal bone resorption was prevented. Significantly, in two cases bone regeneration coronal to the IAJ and onto the abutment surface occurred, completely eliminating the negative sequelae of the IAJ microgap.

In contrast, abutments devoid of laser-ablated microgrooved surfaces, exhibited little evidence of robust fibroblastic activity at the abutment-tissue interface. A long JE extended along the abutment and implant collar surfaces, preventing formation of the physiologic connective tissue barrier and causing crestal bone resorption. Parallel rather than functionally oriented perpendicular connective tissue fibers apposed the abutment-implant surfaces.


© Int J Periodontics Restorative Dent, Volume 30, 2010. p. 245-255

S. Weiner, JL Simon, DS Ehrenberg, B Zweig & JL Ricci.

Abstract

Purpose:
The purpose of this study was to examine the crestal bone, connective tissue, and epithelial cell response to a laser microtextured collar compared with a machined collar, in the dog model.

Materials and Methods:
Six mongrel dogs had mandibular premolars and first molars extracted and after healing replaced with BioLok implants 4x8 mm. Each dog had 3 control implants placed on one side of the mandible and 3 experimental, laser microtextured, implants placed contralaterally. After 3 months, 1 dog was killed. Bridges were placed on the implants of 4 of the dogs. The sixth dog served as a negative control for the duration of the experiment. Two of the dogs were killed 3 months after loading, of the dogs were killed 6 months after loading as was the negative (unloaded) control. Histology, electron microscopy, and histomorphometric analysis was done on histologic sections obtained from block sections of the mandible containing the implants.

Results:
Initially the experimental implants showed greater bone attachment along the collar. With time the bone heights along the control and experimental collars were equivalent. However, the controls had more soft tissue downgrowth, greater osteoclastic activity, and increased saucerization compared with sites adjacent to experimental implants. There was closer adaptation of the bone to the laser microtextured collars.

Conclusion:
Use of tissue engineered collars with microgrooving seems to promote bone and soft tissue attachment along the collar and facilitate development of a biological width.

© Implant Dentistry, Volume 17, Number 2, 2008. p. 217-228

GE Pecora, R Ceccarelli, M. Bonelli, H. Alexander & JL Ricci.

Introduction:
A tapered dental implant (Laser-Lok [LL] surface treatment) with a 2 mm wide collar, that has been laser micromachined in the lower 1.5 mm to preferentially accomplish bone and connective tissue attachment while inhibiting epithelial downgrowth, was evaluated in a prospective, controlled, multicenter clinical trial.

Materials :
Data are reported at measurement periods from 1 to 37 months postoperative for 20 pairs of implants in 15 patients. The implants are placed adjacent to machined collar control implants of the same design. Measurement values are reported for bleeding index, plaque index, probing depth, and crestal bone loss.

Results:
No statistical differences are measured for either bleeding or plaque index. At all measurement periods there are significant differences in the probing depths and the crestal bone loss differences are significant after 7 months (P<0.001). At 37 months the mean probing depth is 2.30 mm and the mean crestal bone loss is 0.59 mm for LL versus 3.60 and 1.94 mm, respectively, for control implant. Also, comparing results in the mandible versus those in the maxilla demonstrates a bigger difference (control implant - LL) in the mean in crestal bone loss and probing depth in the maxilla. However, this result was not statistically significant.

Discussion :
The consistent difference in probing depth between LL and control implant demonstrates the formation of a stable soft-tissue seal above the crestal bone. LL limited the crestal bone loss to the 0.59 mm range as opposed to the 1.94 mm crestal bone loss reported for control implant. The LL implant was found to be comparable with the control implant in safety endpoints plaque index and sulcular bleeding index. There is a nonstatistically significant suggestion that the LL crestal bone retention superiority is greater in the maxilla than the mandible.

© Implant Dentistry. Volume 18(1). February 2009. pp. 57-66.

Myron Nevins, Marc L Nevins, Marcelo Camelo, Janie Lee Boyesen & David M. Kim.

Abstract
This human proof-of-principle study was designed to investigate the possibility of achieving a physical connective tissue attachment to the Laser-Lok microchannel collar of a dental implant. Its 2-mm collar has been micromachined to encourage bone and connective tissue attachment while preventing apical migration of the epithelium. Implants were harvested with the surrounding implant soft and hard tissues after 6 months. The histologic investigation was conducted with light microscopy, polarized light, and scanning electron microscopy.

Results:
The implants were osseointegrated with histologic evidence of direct bone contact. There was a connective tissue attachment to the Laser- Lok microchannels. There were no signs of inflammation. The peri-implant tissues consisted of a dense, collagenous lamina propria covered with a stratified, squamous, keratinizing oral epithelium. The latter was continuous with the parakeratinized sulcular epithelium that lined that lateral surface of the peri-implant sulcus. Apically, the sulcular epithelium overlapped the coronal border of the junctional epithelium. The sulcular epithelium was continuous with the junctional epithelium, which provided epithelial union between the implant and the surrounding peri-implant mucosa. Between the apical termination of the junctional epithelium and the alveolar bone crest, connective tissue directly apposed the implant surface.

Light microscopic evaluation of these specimens revealed intimate contact of the junctional epithelial cells with the implant surface. The microgrooved area of the implants was covered with connective tissue. Polarized light microscopy of this area revealed functionally oriented collagen fibers running toward the grooves of the implant surface. SEM of a corresponding area of the specimen confirmed the presence of attached collagen fibers.

All specimens demonstrated a high degree of bone-to-implant contact and intense remodeling activity. In specimens that showed collagen fibers functionally oriented toward the grooves on the implant surface, remodeling of new bone in the coronal direction was observed. SEM revealed sulcular epithelium with the desquamating activity of the cells and the junctional epithelium. It appears that the connective tissue attachment is instrumental in preserving the alveolar bone crest and inhibiting apical migration of the epithelium.

© The International Journal of Periodontics & Restorative Dentistry. Volume 28, Number 2, 2008

Bae HE, Chung MK, Cha IH & Han DH.

Abstract

Purpose:
This animal study has examined the histomorphometric variations between implants with micro-thread (Oneplant by Warantec, 400µm threads on 2mm collar), micro-grooved (Silhouette implant by Bio-Lok, 8 and 12µm grooved 2mm collar) and machined surface collar designs (Avana implant by Osstem, machined 1mm collar).

Materials and Methods:
Mandibular premolars from four mongrel dogs have been removed and left to heal for three months. One of three different implant systems were placed according to the manufacturers' protocol and left submerged for 8 and 12 weeks. These were then harvested for histological examination. All specimens have shown uneventful healing for the duration of the experiment.

Conclusion:
Within the limitations of this study, it can be concluded that implants with micro-grooved collars had the least change in marginal bone levels and the machined collar implants had the most changes. The macro textured surfaces affected soft tissue response.

Results:
A high bone-to-implant percentage of 66.8% (±8.9%) [BioHorizons BIC% = 80.6%] was found in the examined retrieved implants. Some marginal bone resorption was observed in the crestal part of the implants.

© J Korean Acad Prosthodont. 2008 Dec;46(6):602-609.

Shapoff CA, Lahey B, Wasserlauf PA & Kim DM.

Introduction:
This retrospective radiographic study was organized to evaluate the clinical efficacy of implants with Laser-Lok microtexturing (8- and 12-µm grooves). A physical attachment of connective tissue fibers to the Laser-Lok microtexturing on the implant collar has been previously demonstrated using human histology, polarized light microscopy, and scanning electron microscopy. This analysis of 49 implants demonstrated a mean crestal bone loss of 0.44 mm at 2 years postrestoration and 0.46 mm at 3 years. All bone loss was contained within the height of the collar, and no bone loss was evident to the level of the implant threads. The radiographic evaluation of the clinical application of this implant supports previous findings that establishing a biologic seal of connective tissue fibers around a dental implant may be clinically relevant.

© Int J Periodontics Restorative Dent 2010;30:129-137

Degidi M & Piattelli A

Introduction
Tissue response to any implantable device has been found to correlate with a complex combination of material interface parameters based on composition, surface chemistry, and surface microgeometry. The relative contributions of these factors are difficult to assess.

In vitro and in vivo experiments have demonstrated the role of surface microgeometry in tissue-implant surface interaction although no well-defined relationship has been established. The general relationship, as demonstrated by in vivo experiments on metallic and ceramic implants indicates that smooth surfaces promote formation of thick fibrous tissue encapsulation and rough surfaces promote thinner soft tissue encapsulation and more intimate bone integration. Smooth and porous titanium surfaces have also been shown to have different effects on the orientation of fibrous tissue cells in vitro. Surface roughness has been shown to be a factor in tissue integration of implants with hydroxyapatite surfaces, and to alter cell attachment and growth on polymer surfaces roughened by hydrolytic etching. Roughened surfaces have also shown pronounced effects on differentiation and regulatory factor production of bone cells in vitro. Defined surface microgeometries, such as grooved and machined metals and polymer surfaces have been shown to cause cell and ECM orientation in vivo and can be used to encourage or impede epithelial downgrowth in experimental dental implants. Surface texturing has also been shown to adhere fibrin clot matrix more effectively than smooth surfaces, forming a more stable interface during the collagenous matrix contracture that occurs during healing. This is an effect which may be important in determining early events in tissue integration.

It is likely that textured surfaces work on several levels. These surfaces have higher surface areas than smooth surfaces and interdigitate with tissue in such a way as to create a more stable mechanical interface. They may also have significant effects on adhesion of fibrin clot, adhesion of more permanent extracellular matrix components, and long-term interaction of cells at stable interfaces. We have observed that, in the short term, fibrous tissue cells form an earlier and more organized collagenous capsule at smooth interfaces than at textured interfaces. We suggest that textured surfaces have an additional advantage over smooth surfaces. They inhibit colonization by fibroblastic cell types that arrive early in wound healing and encapsulate smooth substrates.

We have investigated (1) the effects of textured surfaces on colony formation by fibroblasts, and (2) the effects of controlled surface microgeometries on fibroblast colonization. Based on these results, we have designed, fabricated, and tested titanium alloy and commercially-pure titanium implants with controlled microgeometries in in vivo models. These experimental surfaces have highly-oriented, consistent microstructures which are applied using computer-controlled laser ablation techniques. The results suggest that controlled surface microgeometry, in specific size ranges, can enhance bone integration and control the local microstructural geometry of attached bone.


Conclusion:
Immediate functional and non-functional loading seems to be a technique that gives satisfactory results in selected cases.

© Bone Engineering (editor: JE Davies). Chapter 25. Published by Em2 Inc., Toronto, Canada. 2000.

John L Ricci & Harold Alexander.

Introduction
The success or failure of bone and soft tissue-fixed medical devices, such as dental and orthopaedic implants, depends on a complex combination of biological and mechanical factors. These factors are intimately associated with the interface between the implant surface and the surrounding tissue, and are largely determined by the composition, surface chemistry, and surface microgeometry of the implant. The relative contributions of these factors are difficult to assess. This study addresses the contribution of surface microtexture, on a controlled level, to tissue integration.

In this paper, we will describe the common machined, blasted, and etched surfaces used on dental and orthopaedic implants, and describe the development and testing of a series of new laser micromachined surfaces for use on these implants. In previous in vitro experiments, we have investigated (1) the effects of textured surfaces on colony formation by fibroblasts, and (2) the effects of controlled surface microgeometries on fibroblast colonization. Based on these results, we have designed, fabricated, and tested laser microgrooved surfaces with features in specific size ranges, which are applied using computer-controlled laser ablation techniques. We have tested these on titanium alloy and commercially-pure titanium implants in in vivo models. These experimental surfaces have highly-organized and oriented microstructures. They can be produced with features in specific size ranges, and can be applied to specific, highly controlled areas of implants. The results suggest that controlled surface microgeometry, in specific size ranges, can enhance bone and soft tissue integration and control the local microstructural geometry of attached tissue. These surfaces are currently being applied to dental implants and tested in human clinical studies.

© Key Engineering Materials, Vols 198-199 (2001) pp. 179-202.
Trans Tech Publications. Switzerland.

Alexander H, Ricci JL & Hrico GJ.

Abstract

This study, analytically, through finite element analysis, predicts the minimization of crestal bone stress resulting from implant collar surface treatment. A tapered dental implant design with Laser-Lok (LL) and without (control, C) laser microgrooving surface treatment are evaluated. The LL implant has the same tapered body design and thread surface treatment as the C implant, but has a 2-mm wide collar that has been laser micromachined with 8 and 12µm grooves in the lower 1.5 mm to enhance tissue attachment. In vivo animal and human studies previously demonstrated decreased crestal bone loss with the LL implant. Axial and side loading with two different collar/bone interfaces (nonbonded and bonded, to simulate the C and LL surfaces, respectively) are considered. For 80 N side load, the maximum crestal bone distortional stress around C is 91.9 MPa, while the maximum crestal bone stress around LL, 22.6 MPa, is significantly lower. Finite element analysis suggests that stress overload may be responsible for the loss of crestal bone. Attaching bone to the collar with LL is predicted to diminish this effect, benefiting crestal bone retention.

© J Biomed Mater Res B Appl Biomater. Accepted: 7 March 2007

JL Ricci, J Charvet, R Sealey, I Biton, WS Green, SA Stuchin & H Alexander.
Presented at the 21st Annual Meeting of the Society for Biomaterials.
March 18-22, 1995. San Francisco, CA.

Abstract

Introduction:
Soft tissue encapsulation of an implant has been found to correlate with the composition, surface chemistry, and surface microgeometry of the implant material. Surface microgeometry (or surface texture) of metal implants in bone has been shown to influence fibrous capsule formation. For example, smooth surfaces induce thicker fibrous capsule formation than roughened surfaces, suggesting that surface microgeometry influences fibrous tissue proliferation. We evaluated the in vitro response of rat tendon fibroblast (RTF) cell colonies, and human implant capsule fibroblast (HICF) cell colonies, from fibrous capsule tissue from around total hip replacement components, to surfaces roughened by blasting techniques, and to controlled surface microgeometries consisting of small square post projections with features from 3 to 12 µm in size.

Materials and Methods:

RTF cells were grown from hind-foot extensor tendons of 14-day-old Sprague Dawley rats. HICF cells were grown from fibrous capsule tissue samples obtained from patients undergoing total hip revision, including removal of an uncemented hip prosthesis. The tissues, obtained from an area near the proximal stem, were grown as explants under sterile conditions to produce stock cell cultures. All cells were grown as stock cultures, mixed with solubilized collagen, and dispensed and polymerized to initiate "dot" cultures, consisting of 2µL dots each containing 20,000 cells, on all experimental surfaces. These cell-collagen dots acted as sources of cell outgrowth to form growing cell colonies. At 4 and 8 days, cell colonies were fixed, stained, and measured for area of growth using a video camera-equipped stereomicroscope connected to a computer image-processing/image-analysis system. Growth of the cell colonies was measured as area or diameter increase between 4 and 8 days. Roughened surfaces were produced by grit-blasting or bead-blasting of polystyrene culture plates. A masked area was used as a smooth control surface. These surfaces consisted of a range of feature sizes depending on blast medium. The media were similar to those used to texture metal orthopaedic implants and produced similar sized features. Controlled microgeometry substrates were molded in solvent-cast polystyrene from templates precision-fabricated using optical lithography methods at the National Nanofabrication Facility at Cornell University. All surfaces were sputter-coated with a 600-Å layer of TiO2 to simulate an orthopaedic titanium alloy implant surface. The controlled microgeometry surfaces consisted of well-characterized, cross-hatched or checkerboard surface patterns consisting of square posts in 3, 6, 10 and 12µm feature sizes.

Results:
All cell colonies showed consistent growth by day 4, with cell outgrowth observed at the periphery of the dot. Randomly oriented cells formed circular colonies on the control surfaces, and the roughened surfaces. The controlled microgeometry surfaces produced colonies with unusual shapes because of surface restriction of direction of growth. On an individual cell level, the cells were observed to orient along surface structures and in grooves between surface structures. On the smallest controlled microgeometries, each cell was observed to attach to the surfaces of several of the square posts. All of the experimental surfaces significantly inhibited cell colony growth in both types of cells. Significant cell growth inhibition was observed on the grit-blasted Ti-coated surface (GB-Ti) compared to the control Ti-coated surface (C-Ti) and the control untreated culture plate (C-p) as shown in Fig. 1 which represents RTF cell growth. The most efficient cell growth inhibition was observed on the 3µm cross-hatched surface (Fig. 2), although all of the cross-hatched surfaces caused significant inhibition of colony growth. Figure 2 also shows RTF cell colony data.

Conclusions:
RTF and HICF cell colonies grown on roughened surfaces and on a series of controlled microgeometries exhibited pronounced inhibition of growth. The surfaces did not increase cell density in the colonies and the effect was not based on increased substrate surface area. The observed results represent the effects of cellular contact guidance - the ability of substrate microgeometry to influence cell orientation and migration - on overall growth of cell colonies. The observed effects of roughened surfaces and microgeometries on fibrous tissue cell growth, in vitro, may be related to the observation that roughened surfaces cause less fibrous encapsulation in vivo. If so, controlled microgeometry surfaces may be used to effectively suppress fibrous encapsulation.
Acknowledgements:
This work was supported by Orthogen Inc., and was aided by a grant from the Orthopaedic Research and Educational Foundation.

© J Oral Implantol 2002;28:2-8

JL Ricci, R Rose, JK Charvet, H Alexander & CS Naiman.
Presented at the Fifth World Biomaterials Congress.
May 29-June 2, 1996. Toronto, Canada.

Abstract
Introduction:
It has long been recognized that implant surface microtexture can influence tissue interaction. In previous studies we have examined the in vitro interaction of connective tissue fibroblasts with a variety of defined surface microgeometries, including microgrooved, roughened and more complex surfaces. In most cases, these surfaces, while having similar composition, have different (and pronounced) effects on the rate and direction of growth of fibroblast cell colonies. The mechanism of surface microgeometry's effect on cell colony growth rate is unknown. This study investigated the effect of defined surface microgeometry on connective tissue cell colony density, cell attachment area (spreading), and cell shape. The results suggest a possible basic mechanism of surface microgeometry control of attached cell growth.

Materials and Methods:
Rat tendon fibroblast (RTF) cells were grown as stock cultures from hindfoot extensor tendons from 14-day-old Sprague-Dawley rats. Second to fourth-passage cells, grown in Dulbecco's Modified Eagle's Medium containing penicillin-streptomycin and 10% fetal bovine serum were used for all experiments. Cell colonies were grown on these surfaces using a "dot" culture model similar to explant culture models. These cells were suspended in solubilized collagen (Vitrogen, Celltrix, Palo Alto, CA) and 2µL droplets containing 20,000 cells each were polymerized on the experimental surfaces, where they acted as sources of radiating cell colony growth. Light microscopy and image analysis methods were used to measure rate and direction of growth as well as cell density (cells/mm2), cell attachment area (µm2), cell orientation (relative to substrate orientation), and cell elongation (eccentricity, the ratio of cell length to cell width). For individual cell measurements, 30 cells of each experimental group were measured. Experimental substrates consisted of solvent-cast polystyrene surfaces, vapor-deposited with 60nm of TiO2, molded from silicon wafer templates produced by optical lithography methods at the National Nanofabrication Facility at Cornell University (Ithaca, NY). Substrates consisted of mirror-smooth surfaces (controls) and square-wave microgrooves with ridges and grooves 1.75, 6.5 and 12µm in size. Results were analyzed for statistical significance using t-tests.

Results:
All three microgrooved surfaces had a pronounced effect on cell colony growth, cell attachment area, cell eccentricity, cell density, and cell orientation (Table 1): they reduced cell colony growth and cell spreading, increased cell eccentricity (elongation), and effectively oriented the cells parallel to the surface. Cell density on all experimental surfaces was reduced relative to controls.

Discussion:
Well-defined surface microgeometries with the tested dimensions are effective at orienting cells, changing cell shape, and reducing cell growth rates. It is well known that attachment-dependent cells must attach and spread to trigger cell division. The present results suggest that the growth inhibition effect demonstrated by these surfaces may be based on reduction of cell spreading by the surface microgrooves. These experiments suggest that observed differences in fibrous encapsulation of smooth vs. microtextured surfaces may be based on direct suppression of fibroblast spreading and growth by microtextures. These microgeometries have potential application as implant surfaces for control of tissue integration.

Acknowledgements:
The SBM-treated surface provided a greater bone-implant contact that a machined surface after 90 days without loading in this model.

This work was funded by Orthogen Corporation through NSF SBIR Phase I award 9160684.

Soboyejo WO, Nemetski B, Allameh S, Marcantonio N, Mercer C & Ricci J.

This paper presents the results of an experimental study of the interactions between MC3T3-E1 (mouse calvarian) cells and textured Ti6Al4V surfaces, including surfaces produced by laser microgrooving; blasting with alumina particles; and polishing. The multiscale interactions between MC3T3-E1 cells and these textured surfaces are studied using a combination of optical scanning transmission electron microscopy and atomic force microscopy. The potential cytotoxic effects of microchemistry on cell-surface interactions also are considered in studies of cell spreading and orientation over 9-day periods. These studies show that cells on microgrooved Ti6Al4V geometries that are 8 or 12 micron deep undergo contact guidance and limited cell spreading. Similar contact guidance is observed on the surfaces of diamond-polished surfaces on which nanoscale grooves are formed due to the scratching that occurs during polishing. In contrast, random cell orientations are observed on alumina-blasted Ti6Al4V surfaces. The possible effects of surface topography are discussed for scar-tissue formation and improved cell-surface integration.

© J Biomed Mater Res. 2002 Oct; 62(1):56-72.

JC Grew, SR Frenkel, E Goldwyn, T Herman & JL Ricci.
Presented at the 24th Annual Meeting of the Society for Biomaterials.
April 22-26, 1998. San Diego, CA.

Abstract
Introduction:
Implant surface geometry and microgeometry affect tissue responses, although the tissue-implant interaction is incompletely characterized. Physical and chemical properties of synthetic substrates affect the morphology, physiology and behavior of cultured cells of various types. Investigators are just now beginning to describe these in vitro effects in detail. Shape, attachment, migration, orientation, and cytoskeletal organization differ between cells cultured on flat substrates and substrates having regular surface features of micrometric dimensions. We studied murine fibroblast shape, orientation, and microfilament and focal adhesion distribution - parameters relevant to contact guidance and to other factors influenced by substrate microgeometry. Microfilament organization reflects cell shape and orientation, but is also important in signal transduction schemes governing cell attachment, mitosis, migration and apoptosis. Microfilament bundles terminate in clusters of actin-associated proteins, adhesion proteins, and protein kinases having signal transduction functions. We employed assays that revealed the distribution of (1) microfilaments/stress fibers; (2) focal adhesion molecules; and (3) phosphotyrosine, the product of the major class of kinases associated with cell attachments.

Methodology:
NIH-3T3 fibroblasts, 3T3-Li fibroblasts (ATCC, Manassas, VA) and MC-3T3 fibroblasts (gift of JP O'Connor) were grown in DMEM with 10% NCS and 1% antibiotics in 24-well plates containing TiO2-coated, microtextured polystyrene inserts. Culture substrates had either 8µm parallel grooves, 12µm parallel grooves, 3x3µm square posts separated by 3µm perpendicular grooves, or no features (controls). Ten thousand cells were seeded into wells containing the inserts and after 1 day, were prepared for scanning electron microscopy (SEM) or stained with rhodamine-phalloidin.

Results:
By day 4, the 3T3 cells had adhered to all substrates, and by day 8 they showed considerable growth in places approaching confluences. There was no predominant orientation or shape in cells grown on control surfaces. Their cytoplasms showed diffuse rhodamine staining; demonstrable stress fibers were absent. Focal adhesions and phosphotyrosine were diffusely distributed. Cells grown on 8 or 12µm grooved substrates were nearly uniformly oriented in the direction of the grooves. Cells cultured on 8µm grooves mostly grew atop the ridges, often bridging the troughs between ridges. Cells cultured on 12µm grooves mostly grew either atop the ridges or within the troughs, only infrequently bridging the troughs between ridges. Some cells demonstrated limited evidence of stress fibers after 8 days in culture on the grooved surfaces. Focal adhesions and phosphotyrosine were limited to areas of cell-substrate contact; portions of cells spanning troughs lacked focal adhesions and phosphotyrosine. Cells grown on the posted surfaces showed orthogonal arrays of microfilaments that conformed to the intersecting troughs between posts; stress fibers, however, were not observed. These cells either rested atop the posts or settled down onto the posts, with the posts apparently displacing cytoplasm and limiting the distribution of microfilament bundles to areas of basal contact. SEM observations confirmed that the posts penetrated the basal cell membrane surface, with the cell contents settling around the posts. Focal attachments and phosphotyrosine were similarly distributed in these cultures.

Discussion:
This experiment demonstrated that parallel and intersecting grooves are capable of affecting the shape, orientation, cytoskeletal organization, and distribution of focal adhesions in 3T3 fibroblasts, extending our previous findings of these effects in rat tendon fibroblasts. The role of extracellular matrix in guiding this process, while not characterized, is not discounted. The limitation of kinase activity by physical substrate features is a novel finding and could shed light on mechanisms by which cell types respond differentially to substrates. Ultimately, we hope to uncover phenotypic differences in these properties between cell types that will direct the tissue response to implants in ways that improve the incorporation of and extend the functional life spans of the implants.

Acknowledgements:
This work was aided by National Science Foundation SBIR phase I grant #9160684 and by Jersey City State College SBR grant #220251. Microgeometry molds were prepared by the Cornell Nanofabrication Facility.

JC Grew & JL Ricci.
Presented at the Sixth World Biomaterials Congress. Kamuela, HI.
May 15-20, 2000.

Introduction:
Implant surface geometry and microgeometry influence tissue responses to implants. The physical and chemical properties of synthetic substrates affect the morphology, physiology and behavior of cultured cells of various types. To date, studies of tissue-implant interaction have emphasized cell attachment, signaling and other cellular response mechanisms. Cellular attributes influenced by micrometric substrate features include cell shape, attachment, migration, orientation, and cytoskeletal organization. We studied three phenotypic variants of a murine fibroblast cell line to explore the influence of substrate microgeometry on cell shape, orientation, and microfilament distribution. Microfilament organization reflects cell shape and orientation, attendant to cell signaling events that also regulate cell attachment, mitosis, migration and apoptosis. Microfilament bundles (stress fibers) terminate at clusters of actin-associated proteins, adhesion molecules, and protein kinases which contribute to in vitro cell responses to culture substrates.

Methodology:
NIH-3T3 fibroblasts, 3T3-Li fibroblasts (ATCC, Manassas, VA) and MC-3T3 fibroblasts (gift of JP O'Connor) were grown in DMEM with 10% NCS and 1% antibiotics in 24-well plates containing TiO2-coated, microtextured polystyrene inserts. Culture substrates had either 8µm parallel grooves, 12µm parallel grooves, 3x3µm square posts separated by 3µm perpendicular grooves, or no features (controls). Ten thousand cells were seeded into wells containing the inserts and after 1 day, were prepared for scanning electron microscopy (SEM) or stained with rhodamine-phalloidin.

Results:
All three variants of 3T3 fibroblasts adhered to all substrates within 1 day. There was no predominant orientation or shape in cells grown on control surfaces. The cytoplasms of some cells grown on control surfaces showed random arrays of stress fiber, apparently terminating at focal adhesions. Nearly all cells of all types grown on 8 or 12µm grooved substrates were elongated and oriented parallel to the grooves, growing atop the ridges or within the troughs (Figure 1). Cells cultured on 8µm grooves bridged grooves more frequently than cells cultured on 12µm grooves. Few cells of any type demonstrated evidence of stress fibers formation after 1 day in culture on grooved surfaces. Many cells grown on posted substrates displayed stress fibers terminating on posts. These assumed a stellate conformation, with process extending orthogonally from a central cytoplasmic mass and terminating atop the elevated posts (Figure 1). This finding is similar to our previous observations of NIH-3T3 cells grown on posted substrates. SEM observations confirmed the shape and orientation effects of the substrates on the 3T3 variants.

Discussion:
This experiment demonstrated that parallel and intersecting grooves dictate the cell shape, orientation and cytoskeletal organization of three phenotypic variants of 3T3 fibroblasts. The NIH-3T3 variant is a fibrogenic line, while the 3T3-L1 variant is lipogenic and the MC-3T3 variant is osteogenic. The phenotypes of these cells were assessed by alkaline phosphatase assay (MC-3T3 cells are alkaline phosphatase positive) and by Sudan Black B staining (for lipid inclusions in 3T3-Li cells). The roles of extracellular matrix and cell adhesion molecules in the contact guidance events described above were not characterized, but are not discounted. We have previously demonstrated that integrin distribution and tyrosine kinase activity is physically constrained by micrometric substrate features. We hypothesize that the same constraint has occurred in the cells described herein. Elucidation of phenotypic differences between cell types that direct the tissue response to implants may yield information leading to improved implant integration and extended implant lifespan.

Acknowledgements:
This work was aided by NSF grants SBIR-9160684 and DUE-9750533, and by NJCU SBR grant 220253. Microgeometry molds were prepared by the Cornell Nanofabrication Facility.

CL Ware, JL Simon & JL Ricci. Presented at the 28th Annual Meeting of the Society for Biomaterials. April 24-27, 2002. Tampa, FL.

Abstract

Introduction:
This report describes the use of laser-microtextured transcutaneous implants in a rabbit calvarial model to enhance soft tissue integration. Dental and orthopaedic implants are routinely microtextured to enhance tissue integration. Computer-controlled laser microtexturing techniques that produce microgrooved surfaces with defined 8-12µm features on controlled regions of implant surfaces have been developed based on results from cell culture experiments and in vivo models. These textures have been replicated onto the collars of dental implants to provide specific areas for both osteointegration and the formation of a stable soft tissue-implant interface. The objective of this study is to evaluate these implants in a transcutaneous rabbit calvarial model to determine whether controlled laser microtexturing can be used to create a stable interface with connective tissue and epithelium.

Methods:
Laser microtextures were produced on the 4mm diameter collars of modified dental implants designed for rabbit studies (Figure 1). The implants were 4.5mm in length and the threaded portion was 3.75mm in diameter. Implants were produced and supplied by Orthogen Corporation (Springfield, NJ) and BioLok International (Deerfield Beach, FL). The implant surfaces were modified by ablation of defined areas, using an Excimer laser and large-area masking techniques. Controlled laser ablation allows accurate fabrication of defined surface microstructure with resolution in the micron scale range. Laser machined surfaces contained 8µm and 12µm microgrooved systems oriented circumferentially on the collars. The collars of the control implants were "as machined", and were characterized by small machining marks on their surfaces. All implants were cleaned and passivated in nitric acid prior to sterilization.

Four transcutaneous implants were surgically implanted bilaterally in the parietal bones in each rabbit using single-stage procedures. The surgical protocol was similar to dental implant placement. An incision was made over the sagittal suture, and the skin and soft tissues were reflected laterally. Implants were placed using pilot drills and fluted spade drills to produce 3.4mm sites for the 3.75mm diameter implants. The implants were placed with the threaded portion in bone, and the laser-microtextured collar penetrating the subcutaneous soft tissue and epithelium. Each rabbit received two implants on either side of the midline (1 control and 3 experimental implants per subject). The skin was then sutured over the implants. Punch openings were made to expose the tops of the platforms of the implants, and the cover screws were used to fasten down small plastic washers coated with triple antibiotic ointment. The plastic washers were used to prevent the skin from closing over the implant during the swelling that occurred during early healing. They were removed after two weeks. Twelve rabbits were used in the study. Rabbits were sacrificed at 2, 4 and 8 weeks, and the implants and surrounding tissues were processed for histology. Hard and soft tissue response to the implants was examined histologically.

Results and Discussion:
No complications or infections were encountered during the course of the experiment. The 2 and 4 week histology displayed immature soft tissue formations around all implants, and little epithelial interaction with the implant surfaces was noted as the epithelium had not regenerated at the implant surface by 2 weeks, and no clear relationship between epithelium and implant was seen at 4 weeks. 8-week samples showed more mature soft tissue and epithelial tissue. In these samples, the epithelium had fully regenerated and the soft tissue showed more mature and organized collagen. In the control samples, the epithelium consistently grew down the interface between implant and soft tissue and formed a deep sulcus along the implant collar. This sulcus extended to the bone surface and there was little or no direct soft tissue interaction or integration with the control surfaces. The 8-week laser machined implants produced a different pattern of tissue interaction. The epithelium also produced a sulcus at the upper collars of these implants. However, in most cases the sulcus did not extend down as far as the bone surface, but ended at a 300-700µm wide band of tissue, which was attached to the base of the microtextured collar. Even though the laser-microtexturing extended to the top of the collar, this soft tissue attachment formed only at the lower portion of the implant collar, where a stable "corner" of soft tissue attached to both the implant collar and the bone surface. This arrangement of sulcus, epithelial attachment, and soft tissue attachment was similar to the "biologic width" structural arrangement that has been described around teeth and in some cases around implants.

Conclusion:
This preliminary study suggests that laser microtextured surfaces can be applied to transcutaneous implants and used to improve soft tissue integration. Results suggested that the soft tissues at the skin interface are capable of producing an arrangement similar to the "biologic width" arrangement seen around teeth. These laser-machined microtextures are hypothesized to work by increasing surface and organization of attached cells and tissues. They can be used to form a functionally stable interface with soft tissues, establishing an effective transcutaneous barrier. While longer-term studies are needed, the results suggest that performance of transcutaneous prosthetic fixation may be enhanced through the use of regional organized microtexturing.

Frenkel SR, Simon J, Alexander H, Dennis M & Ricci JL.

Abstract


Orthopedic implants often loosen due to the invasion of fibrous tissue. The aim of this study was to devise a novel implant surface that would speed healing adjacent to the surface, and create a stable interface for bone integration, by using a chemoattractant for bone precursor cells, and by controlling tissue migration at implant surfaces via specific surface microgeometry design. Experimental surfaces were tested in a canine implantable chamber that simulates the intramedullary bone response around total joint implants. Titanium and alloy surfaces were prepared with specific microgeometries, designed to optimize tissue attachment and control fibrous encapsulation. TGFβ, a mitogen and chemoattractant (Hunziker EB, Rosenberg LC. J Bone Joint Surg Am 1996;78:721-733) for osteoprogenitor cells, was used to recruit progenitor cells to the implant surface and to enhance their proliferation. Calcium sulfate hemihydrate (CS) was the delivery vehicle for TGFβ; CS resorbs rapidly and appears to be osteoconductive. Animals were sacrificed at 6 and 12 weeks postoperatively. Results indicated that TGFβ can be reliably released in an active form from a calcium sulfate carrier in vivo. The growth factor had a significant effect on bone ingrowth into implant channels at an early time period, although this effect was not seen with higher doses at later periods. Adjustment of dosage should render TGFβ more potent at later time periods. Calcium sulfate treatment without TGFβ resulted in a significant increase in bone ingrowth throughout the 12-week time period studied. Bone response to the microgrooved surfaces was dramatic, causing greater ingrowth in 9 of the 12 experimental conditions. Microgrooves also enhanced the mechanical strength of CS-coated specimens. The grooved surface was able to control the direction of ingrowth. This surface treatment may result in a clinically valuable implant design to induce rapid ingrowth and a strong bone-implant interface, contributing to implant longevity.

© J Biomed Mater Res. 2002;63(6):706-13.

I Giovanna, G Pecora, A Scarano, V Perrotti & A Piattelli.

Abstract


Objective:
To perform a histologic and histomorphometric analysis of the peri-implant tissue reactions and bone-titanium interface in 3 immediately loaded (provisional loaded) titanium implants retrieved from a man after a loading period of 4 months.

Materials and Methods:
A 35 year-old patient with a maxillary partial edentulism did not want to wear a provisional removable prosthesis during the healing period. It was decided to insert 3 definitive implants and use 3 provisional implants for the transitional period. The provisional implants were loaded the same day with a resin prosthesis in occlusal contact. During the second surgical phase, after 4 months, the provisional prosthesis was removed, and the provisional implants were retrieved with a trephine bur. Before retrieval, all implants appeared to be clinically osseointegrated. The specimens were processed for observation under light microscopy.

Results:
At low magnification, it was possible to observe that bone trabeculae were present around the implant. Areas of bone remodeling and haversian systems were present near the implant surface. Under polarized-light microscopy, it was possible to observe that in the coronal aspect of the thread, the lamellar bone showed lamellae that tended to run parallel to the implant surface, while in the inferior aspect of the thread, the bone lamellae ran perpendicular to the implant surface.

© Implant Dentistry. Vol 15, Number 3, 2006.

Giovanna Iezzi, Elisabetta Fiera, Antonio Scarano, Gabriele Pecora & Adriano Piattelli.

Abstract

Little is known about the in vivo healing processes at the interface of implants placed in different grafting materials. For optimal sinus augmentation, a bone graft substitute that can regenerate high-quality bone and enable the osseointegration of load-bearing titanium implants is needed in clinical practice. Calcium sulphate (CaS) is one of the oldest biomaterials used in medicine, but few studies have addressed its use as a sinus augmentation material in conjunction with simultaneous implant placement. The aim of the present study was to histologically evaluate an immediately loaded provisional implant retrieved 7 months after simultaneous placement in a human sinus grafted with CaS. During retrieval, bone detached partially from one of the implants which precluded its use for histologic analysis. The second implant was completely surrounded by native and newly formed bone, and it underwent histologic evaluation. Lamellar bone, with small osteocyte lacunae, was present and in contact with the implant surface. No gaps, epithelial cells, or connective tissues were present at the bone-implant interface. No residual CaS was present. Bone-implant contact percentage was 55% ± 8%. Of this percentage, 40% was represented by native bone and 15% by newly formed bone. CaS showed complete resorption and new bone formation in the maxillary sinus; this bone was found to be in close contact with the implant surface after immediate loading.


© Implant Dentistry. Vol 15, Number 3, 2006.

Stuart J. Froum, Sang-Choon Cho, Helena Francisco, Young-Sang Park, Nicolas Elian & Dennis Tarnow.

Abstract

Endosseous dental implants have traditionally been placed using a two-stage surgical procedure with a 6 to 12 month healing period following tooth extraction. In order to decrease healing time, protocols were introduced which included immediate implant placement and provisionalization following tooth extraction. Although survival rates of implants placed with this protocol are high, postoperative gingival shrinkage and bone resorption in the aesthetic zone are potential limitations when using this technique. The two case reports described herein present a surgical technique for the preservation of anterior aesthetics that combines minimally invasive extraction, immediate implant placement, provisionalization, and the use of implants with a laser micro-grooved coronal design.


© Practical Procedures & Aesthetic Dentistry. Volume 19. No. 10. 2007.