**1. Introduction**

The medical and dental professions, with few exceptions, adapted commercially available tools for use that were developed for drilling other materials [1]. For example, bone-cutting tools, which are largely predicated on the design of metal-cutting instruments. Metal drills are end-cutting tools, e.g., only the tip of the drill is engaged in producing a hole, and the same is true for the vast majority of

bone-cutting drills [2]. Metal drills and most bone drills are also designed to cut at a high rotational velocity, which means that the drill can be advanced with minimal axial thrust force [3]. Metal and bone drills generally have a relatively small rake angle, which means that particles generated by cutting are typically scattered from the site to avoid obstructing the drill. Metal drilling typically requires a lubricant that serves as a coolant [4]; in bone cutting, these functions are replaced by saline irrigation [5].

We studied the biological responses to osteotomy site preparation in multiple animal species [6–9] including humans [10], and these analyses, coupled with computational and finite element modeling [5], prompted us to reconsider the design of a bone-cutting tool, optimized for osteotomy site preparation. The resulting tool, called the OsseoShaper, is designed to limit osteocyte death caused by mechanical and thermal damage, and simultaneously retain osseous coagulum/bone chips generated by bone cutting. For this study, cutting tools were downscaled to accommodate the smaller size of the rat maxillae; however, the ratio of cutting-tool diameter and bone surface area was representative of what is used clinically. The purpose of this study was then to compare osteotomies produced by a downscaled OsseoShaper versus a conventional drill in terms of heat generation, osteocyte viability, bone remodeling, and onset of new bone formation.

#### **2. Materials and Methods**

#### *2.1. Animals and Experimental Plan*

Stanford APLAC approved all procedures (#13146), which conform to ARRIVE guidelines. In total, 18 female Wistar rats (Charles River Laboratories) were used in this study. All animals underwent ovariectomy (OVX) and bilateral maxillary first molar (M1) extraction when they were seven weeks old. Animals were then maintained for eight weeks, during which time the osteoporotic phenotype developed [11,12] and the extraction socket completed its healing [9]. All animals were then subjected to bilateral osteotomy site preparation in the healed M1 location. Animals were sacrificed at intervals of 0.5 days, three days, and seven days. Before surgery, general anesthesia was reached via intraperitoneal injection of ketamine (100 mg/kg) (Vedco, Inc., St. Joseph, MO, USA) and xylazine (10 mg/kg) (Akorn, Inc., Lake Forest, IL, USA), while analgesia was reached via subcutaneous injection of Buprenorphine SR (0.5 mg/kg). After surgery, rats recovered in a controlled, 37 ◦C environment, fed a soft food diet for the duration of the experiment and housed in groups of two. Weight changes were <10%. No adverse events (e.g., uncontrolled pain, infection, prolonged inflammation) were encountered.

#### *2.2. Ovariectomy and Tooth Extraction*

To align our experimental model with the average patient receiving a dental implant, e.g., >50 years old [13], seven-week-old female rats underwent OVX [14]. This produced in our animal model an osteopenic/osteoporotic phenotype, which is representative of patients over 50 years of age [15]. In brief, a dorsal midline incision was made between the mid-back and tail base. The peritoneal cavity was accessed through bilateral muscle layer incisions, the ovary was identified, and the connection between the fallopian tube and the uterine horn was suture-ligated. After bilateral removal of the ovaries, the wounds were closed layer by layer.

In parallel with the OVX, bilateral maxillary first molars (M1) were extracted. This further aligned our experimental model with patients, in which the majority of dental implants are placed in healed extraction sites [16]. In brief, micro-forceps were used to loosen and remove the tooth in toto. Bleeding was controlled by local compression. Healing of the extraction site was confirmed using histology and micro-computed tomographic (μCT) imaging. By post-extraction day 21 (PED21), the extraction site was fully healed, as shown by the fact that the bone volume/total volume (BV/TV) of the extraction site was equivalent to adjacent, pristine bone [10].

#### *2.3. Osteotomy Site Preparation*

To directly compare two surgical drilling tools for their ability to maintain osteotomy site viability, rats were anesthetized before a full thickness periosteal flap was elevated at the M1 tooth extraction site. A handpiece (KaVo Dental, Uxbridge, UK) with saline irrigation was used to produce a pilot 1.0-mm drill hole, followed by step-wise enlargement using progressively larger drill diameters (Table 1). In the OsseoShaper protocol, the same type of pilot drill was used to produce a pilot osteotomy; thereafter, a downscaled prototype of the OsseoShaper was used to enlarge the osteotomy to the same final maximum diameter as was achieved with the conventional surgical drill protocol. The mini OsseoShaper was used without irrigation. Drill speeds were adjusted to result in the same radial velocity for all drills and to compensate for slightly different diameters. Each osteotomy was made with a new drill. After osteotomy, tension-free primary closure of the periosteal flap was achieved using tissue glue (VetClose, Henry Schein, Dublin, OH, USA).



#### *2.4. Tissue Collection and Processing*

Tissues were collected at post-osteotomy day (POD) 0.5 to evaluate micro-damage and programmed cell death caused by surgical drilling, as well as at POD3 and POD7, when new bone formation is initiated [7]. In brief, animals were euthanized; then, the entire maxillae were dissected free from other tissues and transferred to 4% paraformaldehyde (PFA) and stored at 4 ◦C overnight. After fixation, samples were decalcified in ethylenediaminetetraacetic acid (EDTA), embedded in paraffin, and sectioned at an 8-μm thickness for analyses. Tissue sections were deparaffinized in Citrisolv (Decon Labs, Inc., King of Prussia, PA, USA) and hydrated via a series of decreasing concentrations of ethanol before staining or other histological/cellular activity analyses.

#### *2.5. Histology*

For aniline blue staining, sections were treated with a saturated solution of picric acid followed by a 5% phosphotungstic acid solution and staining in 1% aniline blue. Slides were then dehydrated and mounted using Permount (Fisher Scientific, Hampton, NH, USA). For pentachrome staining, sections were pre-treated with 6% nitric acid and stained with toluidine blue solution for 5 min (0.5 g toluidine blue in 100 mL of distilled water at pH 1 to 1.5, adjusted with 0.5% HCl). Picrosirius red staining [17] was used to detect collagenous osteoid matrix. Tissues were stained with picrosirius red then viewed under polarized light. Tightly aligned fibrillary collagen molecules appear red compared to less organized collagen fibrils that show a color of shorter (green–yellow) wavelengths.

#### *2.6. Quantification of Programmed Cell Death*

Terminal deoxynucleotidyl transferase deoxyuridine triphosphate (dUTP) nick end labeling (TUNEL) staining (Roche Diagnostics GmbH, Mannheim, Germany) was performed according to the manufacturers' guidelines. Following deparaffinization and rehydration, paraffin sections were stained by incubating slides in permeabilization solution for 8 min, adding TUNEL reaction mixture, then incubating at 37 ◦C in the dark. Between these steps, paraffin sections were washed with phosphate-buffered saline (PBS). To quantify the extent of apoptotic osteocytes, TUNEL-stained tissue

sections from 4–6 different samples were analyzed. Each section was photographed using a Leica digital image system at 20× magnification. The number of TUNEL-labeled osteocytes corresponding to apoptotic cells was determined, and the cells grouped according to their distance from the cut edge. The corresponding area for each group was then calculated. The number of apoptotic cells per unit area was calculated by dividing the number of apoptotic cells to the corresponding area (cell/mm2).

### *2.7. Tartrate-Resistant Acid Phosphatase (TRAP) Activity*

Identification of osteoclasts was done using TRAP staining. TRAP activity was observed using a leukocyte acid phosphatase staining kit (catalog #386A-1KT, Sigma-Aldrich, St. Louis, MO, USA). Tissue sections were processed according to the manufacturer's instructions. To quantify the TRAP activity, TRAP-stained tissue sections were photographed using a Leica digital image system at 10× magnification. The TRAP+ve area corresponding to osteoclasts was determined within the radial zone extending 300 μm from the cutting edge. The TRAP+ve ratio was calculated by dividing the TRAP+ve pixels by the total pixels of the region of interest.

#### *2.8. Immunostaining*

To localize, within the osteotomies, cells that had initiated differentiation down an osteogenic lineage, immunostaining was performed using standard procedures [18]. In brief, following deparaffinization, endogenous peroxidase activity was quenched by 3% hydrogen peroxide for 5 min, and then washed in PBS. Slides were blocked with 5% goat serum (Vector S-1000) for 1 h at room temperature. The appropriate primary antibody was added and incubated overnight at 4 ◦C, then washed in PBS. The primary antibodies used in this study were Osterix (1:1200; ab22552, Abcam, Cambridge, MA, USA) and Cathepsin K (1:200; ab19027, Abcam, Cambridge, MA, USA). Samples were incubated with appropriate biotinylated secondary antibodies (Vector BA-x) for 30 min, then washed in PBS. An avidin/biotinylated enzyme complex (Kit ABC Peroxidase Standard Vectastain PK-4000, Vectorlabs, Burlingame, CA, USA) was added and incubated for 30 min, and a 3,3 -diaminobenzidine (DAB) substrate kit (Kit Vector Peroxidase substrate DAB SK-4100, Vectorlabs, Burlingame, CA, USA) was used to develop the color reaction. Phalloidin immunostaining was performed using Palloidin Control, DyLight 488 conjugate (1:300; PI21833, Invitrogen, Grand Island, NY, USA).

#### *2.9. Histomorphometric Analyses*

Histomorphometric measurements were performed using ImageJ software v.1.51 (NIH, Bethesda, MD, USA). To quantify the amount of new bone formation in the osteotomy site as a function of time, a minimum of four osteotomy sites were analyzed. For each osteotomy site, a minimum of six aniline blue-stained histologic sections that spanned the distance from the furcation to the apex were used to quantify new bone formation. Each section was photographed using a Leica digital image system at <sup>20</sup>× magnification. To calculate the percentage of new bone formation, the number of aniline blue+ve pixels within an osteotomy was measured and divided by the number of the total pixels in the same osteotomy area.

#### *2.10. Micro-Computed Tomography (μCT)*

Scanning and analyses followed published guidelines [19]. Three-dimensional μCT imaging was performed at various times after surgery. In brief, samples were fixed in 4% PFA at 4 ◦C overnight. Then, they were transferred to 70% ethanol solution for μCT scanning before the decalcification process. A μCT tomography data-acquisition system (VivaCT 40, Scanco, Brüttisellen, Switzerland) at 10.5-μm voxel size (70 kV, 115 μA, 300 ms of integration time) was used for scanning and reconstruction. Bone morphometry was performed using the acquisition system's analysis software (Scanco). Multiplanar reconstruction and volume rendering were carried out using Avizo (FEI, Hillsboro, OR, USA) and ImageJ v1.51 (NIH, Bethesda, MD, USA) software, before being imported into Adobe Photoshop and Illustrator (CC2017, Adobe, San Jose, CA, USA).

#### *2.11. Calculation of Osteotomy Surface Roughness*

To calculate the irregularity of the osteotomy walls, the Shape Filter plugin for ImageJ was employed [20]. Ten transverse sections were used to outline the contours of osteotomies using ImageJ. The contours were then converted to black-and-white images, and the plugin was used to obtain the convexity and solidity values. Convexity measured the surface roughness of a two-dimensional (2D) shape and was defined as H/P, where H was the perimeter of convex hull of the shape, and P was the perimeter of the contour. Solidity was defined as C/A, where C was the area occupied by the contour, and A was the area occupied by the convex hull of the contour. The perimeter of the contour was defined as the total length of the shape's perimeter. Shapes such as a square have equal lengths of convex hulls and perimeter of the contours, which results in a convexity = 1. A star, however, has a pentagon convex hull (consider the shape when surrounded by a rubber band) while the perimeter of the contour is the star shape itself. Since the perimeter of the star contour (P) is larger than the convex hull (H), its convexity (H/P) is <1 and, therefore, its surface is rougher compared to a same-sized pentagon.

#### *2.12. Heat Transfer During Drilling*

The temperature produced when cutting with a conventional protocol involving multiple drills was compared to site preparation with the mini OsseoShaper. Sawbones 35 (Pacific Research Laboratory, Vashon Island, DC, USA) was used. Drills and drilling protocols used are as listed in Table 1. Thermal radiation was measured immediately after drilling via an infrared camera (SEEK CompactPRO, Seek Thermal Application, Santa Barbara, CA, USA). The drilling protocol was repeated six times in new Sawbones. Means and standard deviations were reported.

The temperature distribution during drilling was also calculated in MATLAB using a finite difference method. Details of the heat transfer model are described in Reference [5]. The differences between the conventional drill and mini OsseoShaper models can be summarized as follows: in the conventional high-speed drilling, the heat flux was applied to the drill hole's boundary where the tip of the drill was located, and the tip was moved vertically. Below the drill tip, the value of the heat flux was set to zero, and irrigation was applied above the drill tip. In the OsseoShaper low-speed drilling, the heat flux was applied to the drill hole's boundary at and above the tip due to the tapered shape of the drill, such that the points of engagement between the drill and the bone increased over time as the drill was moved vertically.

#### *2.13. Statistical Analyses*

Results were presented in the form of means ± standard deviations, with *N* equal to the number of samples analyzed. Student's *t*-tests were performed. Significance was set at *p* < 0.05, and all statistical analyses were performed with SPSS software (IBM, Armonk, NY, USA).

#### **3. Results**

#### *3.1. A New Surgical Drilling Tool That Cuts Efficiently at Very Low Speeds*

Most osteotomies are produced through the stepwise enlargement of an initial pilot drill hole with sequentially larger diameter drills [21], all coupled with the use of copious irrigation [22]. We recapitulated that clinical scenario in a rat model, by producing osteotomies using surgical drills with progressively larger diameters. The final drill was 1.6 mm in diameter and was run at 1000 rpm with irrigation (Figure 1A). In osteotomies produced with the downscaled prototype of OsseoShaper, the same pilot drill hole was produced and then followed by a single drill, the OsseoShaper (Figure 1A). The OsseoShaper was run at 50 rpm without irrigation.

**Figure 1.** Osteotomy site preparation with OsseoShaper requires fewer steps and, unlike conventional drills, produces a rough surface. (**A**) All osteotomy site preparations began with the use of a 1.0-mm pilot drill run at 1600 rpm plus irrigation; afterward, the conventional osteotomy procedure used a 1.3-mm drill (1250 rpm plus irrigation) followed by a 1.6-mm drill (1000 rpm plus irrigation). The OsseoShaper protocol used the same 1.0-mm pilot drill at 1600 rpm plus irrigation, and was then followed by the OsseoShaper run at 50 rpm without irrigation. Using a conventional drill (**B**) in plexiglass demonstrates the shape and texture of a cut surfaces, and (**C**) in bone, μCT sections illustrate the parallel walls of the osteotomy. (**D**) Picrosirius red staining of a representative transverse tissue section demonstrates the resulting smooth cut surface. (**E**) Quantification of surface texture, as expressed by convexity and solidity, resulting from a conventional drilling protocol. Using an OsseoShaper (**F**) in plexiglass demonstrates a tapered shape with a threaded surface, (**G**) which is validated by μCT imaging. (**H**) Picrosirius red staining of a representative transverse tissue section demonstrates the textured cut surface and the retention of collagen containing osseous coagulum. Solid and dotted lines show the edge of the osteotomy. Two asterisks indicate *p* < 0.01. Three asterisks indicate *p* < 0.001. Scale bars (**B**,**C**,**F**,**G**) = 1 mm, and (**D**,**H**) = 200 μm. Abbreviations: ab, alveolar bone; os, osteotomy.

A conventional surgical drill is designed to cut only at its tip, which produces a smooth-walled osteotomy, visible both in plexiglass (Figure 1B) and μCT section of bone (Figure 1C). Analyses using picrosirius red staining revealed, under polarized light, the collagen organization at the cut edge when a conventional drill was employed (Figure 1D). Quantification of surface texture, as expressed by convexity and solidity, resulting from a conventional drilling protocol demonstrated the smoother cut edge (Figure 1E). By contrast, the OsseoShaper was designed with a cutting flute running its length; this resulted in a heteromorphic, textured osteotomy surface, visible both in plexiglass (Figure 1F) and in μCT (Figure 1G). Picrosirius red staining demonstrates the textured cut surface and the retention of collagen containing osseous coagulum (Figure 1H).

#### *3.2. The OsseoShaper Allows the Retention of Viable, Autologous Bone Chips in the Osteotomy*

Conventional drills have a rake angle that ranges from 0 to approximately 5◦, the consequence of which is the production of small (<30 μm) bone particles. In addition, conventional drills are typically run at rotational velocities of 800 rpm or higher [23]. Finally, conventional drills are designed to rotate in the same direction, regardless of whether they are being advanced or withdrawn from the osteotomy. Collectively, these attributes result in minimal retention of particulate matter, as can be visualized when cutting Sawbone in vitro (Figure 2A). Coupled with irrigation, the majority of bone debris is typically removed from the osteotomy (Figure 2B).

**Figure 2.** OsseoShaper-produced osteotomies retain more viable bone chips and osseous coagulum. Gross view of a hole produced in 0.32 g/cc Sawbone prepared with (**A**) a conventional drilling protocol versus (**C**) OsseoShaper. Representative transverse sections stained with aniline blue in the osteotomy sites using (**B**) a conventional drilling protocol and (**D**) OsseoShaper protocol. Micro-CT imaging of an osteotomy prepared with (**E**) a conventional drilling protocol versus (**G**) an OsseoShaper. (**F**) Quantification of bone chips in the osteotomy by μCT imaging (*N* = 5). Representative tissue sections of bone chips produced by the OsseoShaper using (**H**) 4 ,6-diamidino-2-phenylindole (DAPI) and (**I**) phalloidin staining. Intra-operative view of an osteotomy prepared with conventional drills versus the OsseoShaper in rats (**J**,**M**), in mini-pigs (**K**,**N**), and in patients (**L**,**O**). Arrows indicate the osteotomy. Small arrows in (**D**) indicate the osteoid matrix. Dotted lines show the edge of the osteotomy. Asterisk indicates *p* < 0.05. Scale bars = 1 mm (**A**,**C**,**J**–**O**) and 200 μm (**B**,**E**). Abbreviations: as indicated previously.

By comparison, the rake angle on a mini OsseoShaper produces osseous coagulum and relatively large (~100 μm) bone chips; additionally, the OsseoShaper is designed to be reversed upon removal. These features result in the collection of bone chips in the cutting flutes, which are then transferred into the osteotomy while the tool is being withdrawn. These events can be visualized when cutting Sawbone (Figure 2C), and upon histologic examination of the osteotomy using aniline blue staining to detect osteoid matrix (Figure 2D).

Micro-CT imaging was used to quantify the volume of osseous coagulum and bone chips retained in the osteotomy. These analyses verified that OsseoShaper osteotomies retained significantly more osseous material than did conventionally prepared osteotomies (Figure 2E,F,G). A closer examination of the bone chips produced by the OsseoShaper using 4 ,6-diamidino-2-phenylindole (DAPI; to detect viable cells) and phalloidin staining (to detect actin filaments) revealed that a subset of chips retained viable osteocytes within the osseous matrix (Figure 2H) and that the majority of chips were surrounded by viable cells (Figure 2I). Clinically, bone chips were only visible in the OsseoShaper-prepared osteotomy sites; this aspect was consistent among species, including rats, mini-pigs, and humans (Figure 2J–O).
