*3.3. The Mini OsseoShaper Preserves Peri-Implant Bone by Limiting Heat Transfer and Minimizing Thermal Apoptosis*

A zone of osteocyte death is produced by conventional drilling [6,7,10]. For example, cutting at 1000 rpm with irrigation produced a ~250-μm-wide, circumferential distribution of TUNEL+ve, apoptotic osteocytes (Figure 3A, quantified in C). By comparison, minimal osteocyte apoptosis was detected after OsseoShaper site preparation (Figure 3B, quantified in C).

**Figure 3.** The OsseoShaper generates less heat, which results in a smaller zone of cell death and less tartrate-resistant acid phosphatase (TRAP)-mediated bone remodeling than conventional drills. (**A**) Representative tissue section from an osteotomy prepared using standard drills, where terminal deoxynucleotidyl transferase deoxyuridine triphosphate (dUTP) nick end labeling (TUNEL)+ve cells are apoptotic osteocytes. (**B**) Equivalent tissue section from an osteotomy prepared using the OsseoShaper, where the majority of apoptotic cells are detected in the osseous coagulum. (**C**) Distribution of TUNEL+ve cells as a function of distance from cut edge of osteotomy. Computational models were used to map the distribution of heat in bone as a function of distance from the cut edge, in osteotomies produced using (**D**) conventional drills and (**E**) using the OsseoShaper. (**F**) Calculated temperatures in bone, expressed as a function of radial distance from conventional drills (dotted red line) and from the OsseoShaper (blue line) (*N* = 6). (**G**) Representative transverse tissue section from an osteotomy produced with conventional drills, analyzed on for TRAP activity on post-osteotomy day 7 (POD7). (**H**) TRAP activity on a representative transverse tissue section from an OsseoShaper-produced osteotomy. (**I**) Quantification of TRAP+ve pixels/total pixels in the region of interest (ROI). A dotted line is used to indicate the cut edge of the osteotomy. Asterisk indicates *p* < 0.05. Scale bars = 200 μm. Abbreviations: as indicated previously.

A computational model was used to calculate peak temperatures produced by both types of cutting tools, taking into account the speed at which the drills were run, the density of the bone being cut, and, in the case of conventional drill protocols, the use of irrigation [5]. In the case of conventional protocols, a peak temperature of ~80 ◦C was generated at the cut edge, despite the use of copious irrigation (Figure 3D; quantified in F). Temperatures decreased as a function of distance from the cut edge but nevertheless, temperatures were >40 ◦C within a ~150-μm circumferential zone (Figure 3A and [6]).

By comparison, the mini OsseoShaper generated significantly lower (~40 ◦C) peak temperatures (Figure 3E; quantified in F). Even without the use of irrigation, calculated temperatures immediately adjacent to the cut edge remained in the physiologic range (Figure 3F), well below temperatures known to cause osteocytes necrosis, i.e., 45 ◦C [24].

An in vitro method supported our conclusion that drilling with the mini OsseoShaper produced less heat. Using Sawbones, site preparation was carried out following the same protocol as used for the site preparation in the rat maxilla (Figure 1A) and, immediately thereafter, the temperature of each drill was measured using an infrared camera (Figure S1, Supplementary Materials). The same method was used to measure heat radiating from the mini OsseoShaper. In the conventional protocol, the heat radiating from conventional drills was significantly higher for each step compared to the heat radiating from the mini OsseoShaper (Figure S1).

Osteocyte death is typically accompanied by peri-implant bone resorption [6,7]. In the case of osteotomies produced with conventional drills, the osteoclast marker TRAP was detected throughout the bone adjacent to the osteotomy edge, as well as in the osteotomy itself (Figure 3G). By contrast, mini OsseoShaper osteotomies exhibited minimal TRAP-mediated bone resorption (Figure 3H). The TRAP activity that was detected reflected new bone remodeling in the osteotomy (Figure 3H; quantified in I).

#### *3.4. In Mini OsseoShaper Osteotomies, New Bone Formation Is Accelerated*

The OsseoShaper was designed to retain osseous coagulum, e.g., mineralized particles including cortical and trabecular bone chips, blood, and stroma that have inherent osteogenic potential [25,26]. On POD3, evidence of this retained osseous coagulum was abundant; compared to conventionally prepared osteotomies, those prepared with the mini OsseoShaper were filled with aniline blue+ve osteoid matrix (compare Figure 4A,B). This matrix served as a nidus for new bone formation and remodeling, as demonstrated by significantly higher Cathepsin K (Figure 4C,D; quantified in E) and Osterix (Figure 4F,G) expression in the mini OsseoShaper osteotomies. By POD7, mini OsseoShaper osteotomies were filled with new bone at a time point when conventionally prepared osteotomies had not yet started to repair (Figure 4H,I; quantified in J).

**Figure 4.** OsseoShaper drilling protocol promotes alveolar bone healing. Representative transverse tissue sections stained with aniline blue on post-osteotomy day 3 (POD3) following osteotomy site preparation with (**A**) conventional drills versus (**B**) the Nobel OsseoShaper. Note the presence of osseous coagulum in the osteotomy site prepared with the OsseoShaper. Adjacent tissue sections immunostained with Cathepsin K in the osteotomy sites of (**C**) conventional drills versus (**D**) the Nobel OsseoShaper. (**E**) Quantification of Cathepsin K+ve pixels/total pixels in the osteotomy site. Adjacent tissue sections immunostained with Osterix in the osteotomy sites of (**F**) conventional drills versus (**G**) the OsseoShaper. (**H**) Tissue sections stained with aniline blue show minimal new bone formation in conventional drill group, while (**I**) osteotomies in the OsseoShaper group show more new bone formation on POD7. (**J**) Quantification of aniline blue+ve pixels/total pixels in the osteotomy site. Dotted lines show the edge of the osteotomy. One asterisk indicates *p* < 0.05. Two asterisks indicate *p* < 0.01. Scale bars = 100 μm. Abbreviations: as indicated previously.

#### **4. Discussion**

Most reconstructive surgeries involve the cutting and removal of bone tissue [27] and, ideally, the goal is to resect a well-defined volume of bone and leave behind a cut edge that is favorable to early cell attachment and matrix deposition [28,29]. Clinicians universally agree that the preservation of cell viability is of utmost importance [30–32], and that high-speed rotating instruments can compromise this viability because they create thermal and mechanical trauma [33–37]. Irrigation can reduce some of the heat generated by high-speed rotatory surgical drills [22,38], but irrigation also removes bone chips, connective tissue stroma, blood, and stem-cell populations, collectively referred to as osseous coagulum, which have osteogenic potential [39–42].

The importance of preserving bone viability led to the development of a wide variety of new cutting tools for bone [43]. For example, gas and solid-state lasers use linear thermal absorption to ablate osteoid tissues and, while they are effective at removing the bone, they also generate heat and consequently show many of the same detrimental effects as drilling [44,45]. Plasma ablation lasers avoid some of these problems by creating energy pulses in very small (i.e., μm) zones that result in very high (several thousand Kelvin) temperatures over a very short (picosecond) duration. The result is limited thermal damage to the bone [46]; technical constraints, however, limit the use of these lasers in most clinical practices [47].

The OsseoShaper was designed to efficiently cut bone at a low (<50 rpm) velocity. This low-speed drilling results in less bone being cut per unit time and, therefore, less heat evolution per unit time (Figure 3). Less heat generation by the OsseoShaper translates into less of a temperature rise in the bone, which obviates the need for a coolant (Figure 3). The biological sequelae of lower heat generation by the mini OsseoShaper was shown by analyses for osteocyte apoptosis and osteoclast activity; because of the minimal temperature rise, few osteocytes underwent programmed cell death, which translated into less peri-implant bone resorption (Figure 3). Clinicians are fully aware that a viable osteotomy site is critical for new bone formation, and this point is perhaps best illustrated by the extent to which surgeons will go to reduce heat produced by rotary cutting tools. Here, we show that improved osteotomy site viability is indeed directly related to enhanced osteogenesis, which we believe will logically translate into a faster osseointegration of an implant placed into such osteotomies.

#### *4.1. A Unique Design That Enables Retention of Bone Chips and Osseous Coagulum in an Osteotomy Site*

Most drills produce bone chips and osseous coagulum, which has inherent osteogenic material that can stimulate new bone formation [25,26]. Most of this osteogenic material is flushed out of the site by irrigation [25], which is required to cool conventional drills. The rake angle of the OsseoShaper produces larger bone chips than conventional drilling protocols.

Most conventional drills rotate clockwise, whether advancing or withdrawing the tool and, coupled with the high rotational velocity, effectively disperse the bone chips and osseous coagulum. The OsseoShaper slowly rotates clockwise when advanced and is then reversed upon withdrawal; this design feature effectively retains bone chips and osseous coagulum in the osteotomy site (Figure 2). This feature was also visible in osteotomy site preparation performed in mini-pig and human individuals (Figure 2). Historic studies demonstrated that such bone chips that remain in situ are highly osteogenic [48].

Cutting flute placement affects the roughness of the osteotomy. Compared to the smooth-walled osteotomies produced by conventional drills, osteotomies produced by the OsseoShaper are textured (Figure 1). Some investigators speculated that a textured surface represents an optimal site for new bone deposition because it mimics the bone surface left behind after osteoclast-mediated remodeling [49].

Clinicians recognize that a bone graft from a patient has osteogenic potential and, therefore, a variety of methods were developed in an attempt to collect this autologous material [50]. Most, if not all, of these collection methods necessitate removal of the autologous bone chips from the body and storage ex vivo. In doing so, the bone graft material is potentially subjected to desiccation, temperature changes, e.g., deviations from 37 ◦C, and bacterial contamination. Use of the OsseoShaper negates

these concerns; bone chips remain in situ and, in doing so, their viability is likely to be enhanced and/or preserved.

### *4.2. A Streamlined Protocol for Osteotomy Site Preparation*

In conventional drilling protocols, a pilot hole is first produced; then, the osteotomy is gradually enlarged through the use of progressively larger diameter drills. A pilot hole is also created before use of the OsseoShaper, after which the final sized osteotomy is produced in a single step (Figure 1). In conventional drilling protocols, the use of multiple drills increases the chance of deviating from the intended axis of the osteotomy, which in turn impacts the axis of the implant placed into the osteotomy [51]. By reducing the number of surgical drills required to produce the final osteotomy, the alignment error is also effectively reduced [52], and subsequent implant placement will follow the axis of the last drill.

#### **5. Conclusions**

In our study, we present a new drill design that is meant to efficiently cut bone at a very low rotational speed, obviating the need for irrigation as a coolant and a lubricant. Osteocyte viability is maintained by the low-speed cutting that produces little heat. Autologous bone chips are generated and maintained on site thanks to the lack of irrigation, coupled with the unique design of the cutting flutes. This osseous coagulum has inherent osteogenic capacities. Collectively, a robust formation of new bone is observed with the new drill design, at rates significantly faster than those observed with conventional drilling protocols. These data have practical applications for clinical implant site preparation and alveolar bone reconstruction.

**Supplementary Materials:** The following are available online at http://www.mdpi.com/2077-0383/8/2/170/s1, Figure S1: Thermal radiation measurements of conventional and Osseoshaper protocols in Sawbones.

**Author Contributions:** Conceptualization, S.H. and A.Q.; methodology, J.A.H. and J.B.B.; data generation, C.-H.C., B.R.C., M.A. (Masaki Arioka), B.L., U.S.T., M.A. (Maziar Aghvami), B.S., W.H., A.Q., and O.B.; writing—original draft preparation, C.-H.C., B.R.C., M.A. (Masaki Arioka), J.B.B., and J.A.H.; writing—review and editing, C.-H.C., B.R.C., M.A. (Masaki Arioka), J.B.B., W.H., and J.A.H. All authors read and approved the manuscript.

**Funding:** This work was supported by NIH R01 DE024000-12 to J.A.H. and J.B.B. and a grant from Nobel Biocare Services AG, Kloten, Switzerland (grant number 2015-1400).

**Acknowledgments:** Special thanks to Audrey Schmitt, DDS, MSc (periodontist, private practice, Rouen, France) for providing surgical photographs from a conventional osteotomy site preparation. J.A.H. and J.B.B. are paid consultants for Nobel Biocare.

**Conflicts of Interest:** The authors declare no conflicts of interest.

#### **References**


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