Accepted Manuscript The effect of ultraviolet-mediated photofunctionalization for bone formation around medical titanium mesh. Makoto Hirota , DDS, PhD Takayuki Ikeda , DDS, PhD Masako Tabuchi , DDS, PhD Toshinori Iwai , DDS Iwai Tohnai , DDS, PhD Takahiro Ogawa , DDS, PhD PII:
S0278-2391(14)00557-6
DOI:
10.1016/j.joms.2014.05.012
Reference:
YJOMS 56328
To appear in:
Journal of Oral and Maxillofacial Surgery
Received Date: 8 January 2014 Revised Date:
3 May 2014
Accepted Date: 8 May 2014
Please cite this article as: Hirota M, Ikeda T, Tabuchi M, Iwai T, Tohnai I, Ogawa T, The effect of ultraviolet-mediated photofunctionalization for bone formation around medical titanium mesh., Journal of Oral and Maxillofacial Surgery (2014), doi: 10.1016/j.joms.2014.05.012. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT
The effect of ultraviolet-mediated photofunctionalization for bone formation around medical titanium mesh. Makoto Hirota, DDS, PhD†§1, 2, Takayuki Ikeda, DDS, PhD§1, Masako Tabuchi, DDS,
RI PT
PhD§1, Toshinori Iwai, DDS ƒ2, Iwai Tohnai, DDS, PhD¶2, Takahiro Ogawa, DDS, PhD¶1 1
Laboratory for Bone and Implant Sciences, The Jane and Jerry Weintraub Center for
SC
Reconstructive Biotechnology, Division of Advanced Prosthodontics, Biomaterials and Hospital Dentistry, UCLA School of Dentistry, 10833 Le Conte Avenue, Box 951668,
2
M AN U
Los Angeles, CA 90095-1668, USA
Department of Oral and Maxillofacial Surgery, Yokohama City University Graduate
School of Medicine, 3-9 Fuku-ura, Kanazawa-ku, Yokohama 236-0004, Japan
§ Visiting Scholar ƒ Assistant Professor
EP
¶ Professor
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† Associate Professor
Corresponding author: Makoto Hirota Department of Oral and Maxillofacial Surgery, Yokohama City University Graduate
AC C
2
School of Medicine
3-9 Fuku-ura, Kanazawa-ku, Yokohama 236-0004, Japan TEL: +81-45-787-2659 FAX: +81-45-785-8438 Email: [email protected]
ACCEPTED MANUSCRIPT
The effect of ultraviolet-mediated photofunctionalization for bone formation around medical titanium mesh
RI PT
Makoto Hirota, DDS, PhD†§1, 2, Takayuki Ikeda, DDS, PhD§1, Masako Tabuchi, DDS, PhD§1, Toshinori Iwai, DDS ƒ2, Iwai Tohnai, DDS, PhD¶2, Takahiro Ogawa, DDS, PhD¶1 1
Laboratory for Bone and Implant Sciences, The Jane and Jerry Weintraub Center for Reconstructive Biotechnology, Division of Advanced Prosthodontics, Biomaterials and Hospital Dentistry, UCLA School of Dentistry, 10833 Le Conte Avenue, Box 951668, Los Angeles, CA 90095-1668, USA 2
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Department of Oral and Maxillofacial Surgery, Yokohama City University Graduate School of Medicine, 3-9 Fuku-ura, Kanazawa-ku, Yokohama 236-0004, Japan
M AN U
† Associate Professor § Visiting Scholar ƒ Assistant Professor ¶ Professor
Corresponding author: Makoto Hirota Department of Oral and Maxillofacial Surgery, Yokohama City University Graduate School of Medicine 3-9 Fuku-ura, Kanazawa-ku, Yokohama 236-0004, Japan TEL: +81-45-787-2659 FAX: +81-45-785-8438 Email: [email protected]
AC C
EP
TE D
2
ACCEPTED MANUSCRIPT
ABSTRACT Purpose: The new technology of photofunctionalization with ultraviolet (UV) light for titanium implants has earned considerable attention. We have hypothesized that UV light treatment would enhance bone formation on titanium mesh.
RI PT
Methods: Investigators have implemented in vitro and in vivo experiments to examine the effectiveness of UV treatment for bone formation on titanium mesh surfaces. A
titanium mesh for medical use was prepared as samples, which were autoclaved and
stored under dark ambient conditions for 4 weeks. UV treatment was performed for 12
SC
min. Carbon contamination, hydrophilicity, and protein adhesion of the titanium mesh surface were examined in an in vitro model. Bone tissue formation around the titanium
M AN U
mesh was observed in a rat femur bone model. Mann–Whitney’s U-test was used to examine differences between untreated and UV-treated groups. P-values of 80% (P < .01). The hydrophobic surface of the untreated titanium mesh became superhydrophilic after UV-mediated
TE D
photofunctionalization (P < .01). The amount of protein adsorbed onto titanium was 1.5- to 3-times greater in photofunctionalized titanium mesh surfaces than in untreated titanium mesh surfaces (P < .01). In the animal experiment, the newly formed bone on the UV-treated titanium mesh was approximately 2.5-times greater than that on the
EP
untreated mesh (P < .05).
Conclusion: UV-mediated photofunctionalization is effective, as demonstrated by
AC C
enhanced bone tissue formation on the titanium mesh. Future studies will focus on bone augmentation using an UV-mediated photofunctionalized titanium implant and mesh.
ACCEPTED MANUSCRIPT
INTRODUCTION The new technology of ultraviolet (UV)-mediated photofunctionalization for titanium implants has earned considerable attention. Titanium is an important biomaterial that is widely used in oral and maxillofacial surgery. Recently, studies have
RI PT
revealed that the titanium surface is covered with hydrocarbon after some production processes, such as a machine cut-and-etch treatment 1-3. Hydrocarbon contamination on titanium surfaces causes biological aging of titanium and reduces osteoblast activity on the surface 1-3. It takes 4 weeks for the aging to be accomplished after production or
SC
processing of titanium surfaces, and 4-week-old titanium surfaces appear hydrophobic, with less attachment to proteins and osteoblasts 1-5. To improve aging titanium surfaces,
M AN U
a photofunctionalization technique with UV light has been developed 1-3. The UV treatment, by a combination of UVA and UVC for the aging titanium surface, dramatically changes the surface properties, as expressed by superhydrophilicity, decontamination of accumulated carbon, and positively converted electrostatic charges. These factors act synergistically to increase the recruitment, attachment, and retention of osteogenic cells 1-3.
TE D
Since Boyne et al. 6 first reported the osseous restoration of deficient alveolar ridges using titanium mesh, bone augmentation surgery with the titanium mesh applied to the atrophied alveolar bone before implant therapy has been developed 7. The alveolar ridge bone augmentation technique using the autogenous bone and the titanium mesh is
EP
well established and is a reliable procedure for the restoration of approximately 5 mm to the height of a bone defect around an implant 7-9, indicating that the titanium mesh
AC C
ensures a bone-regenerative environment. A UV-mediated photofunctionalized titanium surface enhanced bone
generation in a bone-healing environment 10, suggesting that photofunctionalized titanium can be used as a material for bone regeneration, i.e., the UV treatment could be effective for the further enhancement of osteoconductivity of the titanium mesh. The osteoconductivity of the titanium mesh and its possible enhancement have not previously been viewed as important biological requirements. The present study describes the effectiveness of the UV treatment of the titanium mesh as an osteoconductive material by analyzing osteoblast activity in vitro and establishing three-dimensional in vivo morphogenetic profiles of the newly formed bone tissues on
ACCEPTED MANUSCRIPT
the surface.
MATERIALS AND METHODS Titanium mesh surface characterization and photofunctionalization
RI PT
A titanium mesh (thickness, 0.2 mm), made of commercially pure grade-2 titanium with anodizing treatment (Synthes K.K., Tokyo, Japan), was trimmed to fit
12-well culture plates for in vitro studies or cut to 3 × 10 mm for in vivo studies. All meshes were autoclaved and stored in a dark room for 4 weeks. The titanium mesh
SC
contained a round hole (diameter, 1.8 mm) within a titanium frame (width, 0.3-0.4 mm), with a calculated hole ratio of 51.0%. The surface morphology and chemistry were examined using a scanning electron microscope (SEM; XL30, Philips, Eindhoven,
M AN U
Netherlands) and energy-dispersive X-ray spectroscopy (EDS), respectively. The hydrophilic and hydrophobic properties of titanium mesh surfaces were evaluated by measuring the contact angle with 5 µL of H2O. For evaluation of the hydrophilicity of mesh surfaces, trimmed meshes were put on a clean, flat table. A 5-µL droplet of H2O was then put on the mesh surface. Photofunctionalization was performed by treating the
TE D
titanium mesh with UV light for 12 min using a photo device (Ushio Inc., Tokyo, Japan) immediately before the in vitro or in vivo experiment. The machine was optimized for UV light efficacy to the titanium surface. Protein adsorption
EP
Bovine serum albumin (BSA; Pierce Biotechnology, Inc., Rockford, IL, USA) and bovine plasma fibronectin (Sigma-Aldrich, St. Louis, MO, USA) were used as
AC C
model proteins. A 300-µL quantity of protein solution (1 mg/mL protein/saline) was pipetted onto and spread over the trimmed titanium mesh that was put into each well of a 12-well plate. The trimmed mesh was not fixed to the well, but remained stationary during the incubation. After either 6 or 24 h of incubation in sterile humidified conditions at 37°C, the solution containing non-adherent proteins was removed and mixed with micro bicinchoninic acid (BCA; Pierce Biotechnology) at 37°C for 60 min. The amount of protein was quantified using a microplate reader at 562 nm.
Animal experiment
ACCEPTED MANUSCRIPT
Eight-week-old male rats (Charles River, San Diego, CA, USA) were anesthetized by inhalation with 1–2% isoflurane. After their legs were shaved and scrubbed with 10% povidone–iodine solution, the distal aspects of the femurs were carefully exposed via a skin incision and muscle separation. The corner angle of the
RI PT
femur was used for titanium mesh placement. The bone defect for implantation of the titanium mesh into the bone marrow cavity was created by the drilling of a rectangular osteotomy (0.25 mm × 10 mm) along the longitudinal axis of the femur by means of a bur and scalpel. An untreated or photofunctionalized titanium mesh was placed in the
SC
bone with passive retention (Figure 1). The surgeon could not be blinded to the material being implanted because the photofunctionalized titanium mesh easily attracted blood in
M AN U
the surgical site because of its hydrophilicity. Muscle and skin were sutured separately with resorbable suture thread. The protocol was approved by the Chancellor’s Animal Research Committee at the University of California at Los Angeles (UCLA), and all experimentation was performed in accordance with the United States Department of Agriculture (USDA) guidelines on animal research.
Morphological and elemental analysis of the titanium mesh–tissue complex in vivo
TE D
Two weeks after surgery, titanium mesh samples were taken from the rat femurs, soaked in agitated H2O for 1 h, and dried under heat and vacuum. After being carbon-sputter-coated, the specimens were examined using SEM. Based on the SEM images, area and thickness of newly formed tissue on titanium mesh surfaces were
EP
quantified with imaging software (Image J; NIH, Bethesda, MD, USA). The elemental composition of the covering tissues and the titanium mesh complex was analyzed by
AC C
EDS.
Three-dimensional micro-CT analysis of newly formed tissue in mesh holes The method for the assessment of bone formation around titanium has been
previously described 11. Briefly, titanium mesh/tissue specimens fixed in 10% buffered formalin were scanned by micro-CT (µCT 40, Scanco Medical, Bassersdorf, Switzerland) with an isotropic resolution of 8 µm. Approximately 1,250 and 300 CT slices were imaged along the longitudinal axis of and parallel to the titanium mesh, respectively, at an X-ray energy level of 70 kVp and a current of 114 µA. The grayscale images were processed using a Gaussian low-pass noise filter and threshold algorithms
ACCEPTED MANUSCRIPT
to distinguish titanium and the mineralized bone from background. The specific thresholds for titanium and the bone tissue were determined by imaging the original materials. To map the bone generation profile around the titanium, bone fill (%) was analyzed by segmentation as below. The round titanium mesh hole was divided into five
RI PT
200-µm concentric segments, with zone 1 being next to the titanium interface (edge) and zone 5 being the farthest, in the center of the hole. Statistical analysis
In vitro studies were performed in triplicate (n = 3). In vivo experiments were
SC
performed in 7 rats (n = 7). Mann–Whitney’s U-test was used to examine differences between the untreated and photofunctionalized groups. P-values of 60% in the photofunctionalized titanium mesh group, whereas that in the untreated titanium mesh group was
S0278-2391(14)00557-6
DOI:
10.1016/j.joms.2014.05.012
Reference:
YJOMS 56328
To appear in:
Journal of Oral and Maxillofacial Surgery
Received Date: 8 January 2014 Revised Date:
3 May 2014
Accepted Date: 8 May 2014
Please cite this article as: Hirota M, Ikeda T, Tabuchi M, Iwai T, Tohnai I, Ogawa T, The effect of ultraviolet-mediated photofunctionalization for bone formation around medical titanium mesh., Journal of Oral and Maxillofacial Surgery (2014), doi: 10.1016/j.joms.2014.05.012. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT
The effect of ultraviolet-mediated photofunctionalization for bone formation around medical titanium mesh. Makoto Hirota, DDS, PhD†§1, 2, Takayuki Ikeda, DDS, PhD§1, Masako Tabuchi, DDS,
RI PT
PhD§1, Toshinori Iwai, DDS ƒ2, Iwai Tohnai, DDS, PhD¶2, Takahiro Ogawa, DDS, PhD¶1 1
Laboratory for Bone and Implant Sciences, The Jane and Jerry Weintraub Center for
SC
Reconstructive Biotechnology, Division of Advanced Prosthodontics, Biomaterials and Hospital Dentistry, UCLA School of Dentistry, 10833 Le Conte Avenue, Box 951668,
2
M AN U
Los Angeles, CA 90095-1668, USA
Department of Oral and Maxillofacial Surgery, Yokohama City University Graduate
School of Medicine, 3-9 Fuku-ura, Kanazawa-ku, Yokohama 236-0004, Japan
§ Visiting Scholar ƒ Assistant Professor
EP
¶ Professor
TE D
† Associate Professor
Corresponding author: Makoto Hirota Department of Oral and Maxillofacial Surgery, Yokohama City University Graduate
AC C
2
School of Medicine
3-9 Fuku-ura, Kanazawa-ku, Yokohama 236-0004, Japan TEL: +81-45-787-2659 FAX: +81-45-785-8438 Email: [email protected]
ACCEPTED MANUSCRIPT
The effect of ultraviolet-mediated photofunctionalization for bone formation around medical titanium mesh
RI PT
Makoto Hirota, DDS, PhD†§1, 2, Takayuki Ikeda, DDS, PhD§1, Masako Tabuchi, DDS, PhD§1, Toshinori Iwai, DDS ƒ2, Iwai Tohnai, DDS, PhD¶2, Takahiro Ogawa, DDS, PhD¶1 1
Laboratory for Bone and Implant Sciences, The Jane and Jerry Weintraub Center for Reconstructive Biotechnology, Division of Advanced Prosthodontics, Biomaterials and Hospital Dentistry, UCLA School of Dentistry, 10833 Le Conte Avenue, Box 951668, Los Angeles, CA 90095-1668, USA 2
SC
Department of Oral and Maxillofacial Surgery, Yokohama City University Graduate School of Medicine, 3-9 Fuku-ura, Kanazawa-ku, Yokohama 236-0004, Japan
M AN U
† Associate Professor § Visiting Scholar ƒ Assistant Professor ¶ Professor
Corresponding author: Makoto Hirota Department of Oral and Maxillofacial Surgery, Yokohama City University Graduate School of Medicine 3-9 Fuku-ura, Kanazawa-ku, Yokohama 236-0004, Japan TEL: +81-45-787-2659 FAX: +81-45-785-8438 Email: [email protected]
AC C
EP
TE D
2
ACCEPTED MANUSCRIPT
ABSTRACT Purpose: The new technology of photofunctionalization with ultraviolet (UV) light for titanium implants has earned considerable attention. We have hypothesized that UV light treatment would enhance bone formation on titanium mesh.
RI PT
Methods: Investigators have implemented in vitro and in vivo experiments to examine the effectiveness of UV treatment for bone formation on titanium mesh surfaces. A
titanium mesh for medical use was prepared as samples, which were autoclaved and
stored under dark ambient conditions for 4 weeks. UV treatment was performed for 12
SC
min. Carbon contamination, hydrophilicity, and protein adhesion of the titanium mesh surface were examined in an in vitro model. Bone tissue formation around the titanium
M AN U
mesh was observed in a rat femur bone model. Mann–Whitney’s U-test was used to examine differences between untreated and UV-treated groups. P-values of 80% (P < .01). The hydrophobic surface of the untreated titanium mesh became superhydrophilic after UV-mediated
TE D
photofunctionalization (P < .01). The amount of protein adsorbed onto titanium was 1.5- to 3-times greater in photofunctionalized titanium mesh surfaces than in untreated titanium mesh surfaces (P < .01). In the animal experiment, the newly formed bone on the UV-treated titanium mesh was approximately 2.5-times greater than that on the
EP
untreated mesh (P < .05).
Conclusion: UV-mediated photofunctionalization is effective, as demonstrated by
AC C
enhanced bone tissue formation on the titanium mesh. Future studies will focus on bone augmentation using an UV-mediated photofunctionalized titanium implant and mesh.
ACCEPTED MANUSCRIPT
INTRODUCTION The new technology of ultraviolet (UV)-mediated photofunctionalization for titanium implants has earned considerable attention. Titanium is an important biomaterial that is widely used in oral and maxillofacial surgery. Recently, studies have
RI PT
revealed that the titanium surface is covered with hydrocarbon after some production processes, such as a machine cut-and-etch treatment 1-3. Hydrocarbon contamination on titanium surfaces causes biological aging of titanium and reduces osteoblast activity on the surface 1-3. It takes 4 weeks for the aging to be accomplished after production or
SC
processing of titanium surfaces, and 4-week-old titanium surfaces appear hydrophobic, with less attachment to proteins and osteoblasts 1-5. To improve aging titanium surfaces,
M AN U
a photofunctionalization technique with UV light has been developed 1-3. The UV treatment, by a combination of UVA and UVC for the aging titanium surface, dramatically changes the surface properties, as expressed by superhydrophilicity, decontamination of accumulated carbon, and positively converted electrostatic charges. These factors act synergistically to increase the recruitment, attachment, and retention of osteogenic cells 1-3.
TE D
Since Boyne et al. 6 first reported the osseous restoration of deficient alveolar ridges using titanium mesh, bone augmentation surgery with the titanium mesh applied to the atrophied alveolar bone before implant therapy has been developed 7. The alveolar ridge bone augmentation technique using the autogenous bone and the titanium mesh is
EP
well established and is a reliable procedure for the restoration of approximately 5 mm to the height of a bone defect around an implant 7-9, indicating that the titanium mesh
AC C
ensures a bone-regenerative environment. A UV-mediated photofunctionalized titanium surface enhanced bone
generation in a bone-healing environment 10, suggesting that photofunctionalized titanium can be used as a material for bone regeneration, i.e., the UV treatment could be effective for the further enhancement of osteoconductivity of the titanium mesh. The osteoconductivity of the titanium mesh and its possible enhancement have not previously been viewed as important biological requirements. The present study describes the effectiveness of the UV treatment of the titanium mesh as an osteoconductive material by analyzing osteoblast activity in vitro and establishing three-dimensional in vivo morphogenetic profiles of the newly formed bone tissues on
ACCEPTED MANUSCRIPT
the surface.
MATERIALS AND METHODS Titanium mesh surface characterization and photofunctionalization
RI PT
A titanium mesh (thickness, 0.2 mm), made of commercially pure grade-2 titanium with anodizing treatment (Synthes K.K., Tokyo, Japan), was trimmed to fit
12-well culture plates for in vitro studies or cut to 3 × 10 mm for in vivo studies. All meshes were autoclaved and stored in a dark room for 4 weeks. The titanium mesh
SC
contained a round hole (diameter, 1.8 mm) within a titanium frame (width, 0.3-0.4 mm), with a calculated hole ratio of 51.0%. The surface morphology and chemistry were examined using a scanning electron microscope (SEM; XL30, Philips, Eindhoven,
M AN U
Netherlands) and energy-dispersive X-ray spectroscopy (EDS), respectively. The hydrophilic and hydrophobic properties of titanium mesh surfaces were evaluated by measuring the contact angle with 5 µL of H2O. For evaluation of the hydrophilicity of mesh surfaces, trimmed meshes were put on a clean, flat table. A 5-µL droplet of H2O was then put on the mesh surface. Photofunctionalization was performed by treating the
TE D
titanium mesh with UV light for 12 min using a photo device (Ushio Inc., Tokyo, Japan) immediately before the in vitro or in vivo experiment. The machine was optimized for UV light efficacy to the titanium surface. Protein adsorption
EP
Bovine serum albumin (BSA; Pierce Biotechnology, Inc., Rockford, IL, USA) and bovine plasma fibronectin (Sigma-Aldrich, St. Louis, MO, USA) were used as
AC C
model proteins. A 300-µL quantity of protein solution (1 mg/mL protein/saline) was pipetted onto and spread over the trimmed titanium mesh that was put into each well of a 12-well plate. The trimmed mesh was not fixed to the well, but remained stationary during the incubation. After either 6 or 24 h of incubation in sterile humidified conditions at 37°C, the solution containing non-adherent proteins was removed and mixed with micro bicinchoninic acid (BCA; Pierce Biotechnology) at 37°C for 60 min. The amount of protein was quantified using a microplate reader at 562 nm.
Animal experiment
ACCEPTED MANUSCRIPT
Eight-week-old male rats (Charles River, San Diego, CA, USA) were anesthetized by inhalation with 1–2% isoflurane. After their legs were shaved and scrubbed with 10% povidone–iodine solution, the distal aspects of the femurs were carefully exposed via a skin incision and muscle separation. The corner angle of the
RI PT
femur was used for titanium mesh placement. The bone defect for implantation of the titanium mesh into the bone marrow cavity was created by the drilling of a rectangular osteotomy (0.25 mm × 10 mm) along the longitudinal axis of the femur by means of a bur and scalpel. An untreated or photofunctionalized titanium mesh was placed in the
SC
bone with passive retention (Figure 1). The surgeon could not be blinded to the material being implanted because the photofunctionalized titanium mesh easily attracted blood in
M AN U
the surgical site because of its hydrophilicity. Muscle and skin were sutured separately with resorbable suture thread. The protocol was approved by the Chancellor’s Animal Research Committee at the University of California at Los Angeles (UCLA), and all experimentation was performed in accordance with the United States Department of Agriculture (USDA) guidelines on animal research.
Morphological and elemental analysis of the titanium mesh–tissue complex in vivo
TE D
Two weeks after surgery, titanium mesh samples were taken from the rat femurs, soaked in agitated H2O for 1 h, and dried under heat and vacuum. After being carbon-sputter-coated, the specimens were examined using SEM. Based on the SEM images, area and thickness of newly formed tissue on titanium mesh surfaces were
EP
quantified with imaging software (Image J; NIH, Bethesda, MD, USA). The elemental composition of the covering tissues and the titanium mesh complex was analyzed by
AC C
EDS.
Three-dimensional micro-CT analysis of newly formed tissue in mesh holes The method for the assessment of bone formation around titanium has been
previously described 11. Briefly, titanium mesh/tissue specimens fixed in 10% buffered formalin were scanned by micro-CT (µCT 40, Scanco Medical, Bassersdorf, Switzerland) with an isotropic resolution of 8 µm. Approximately 1,250 and 300 CT slices were imaged along the longitudinal axis of and parallel to the titanium mesh, respectively, at an X-ray energy level of 70 kVp and a current of 114 µA. The grayscale images were processed using a Gaussian low-pass noise filter and threshold algorithms
ACCEPTED MANUSCRIPT
to distinguish titanium and the mineralized bone from background. The specific thresholds for titanium and the bone tissue were determined by imaging the original materials. To map the bone generation profile around the titanium, bone fill (%) was analyzed by segmentation as below. The round titanium mesh hole was divided into five
RI PT
200-µm concentric segments, with zone 1 being next to the titanium interface (edge) and zone 5 being the farthest, in the center of the hole. Statistical analysis
In vitro studies were performed in triplicate (n = 3). In vivo experiments were
SC
performed in 7 rats (n = 7). Mann–Whitney’s U-test was used to examine differences between the untreated and photofunctionalized groups. P-values of 60% in the photofunctionalized titanium mesh group, whereas that in the untreated titanium mesh group was
