Bone Regeneration in Osteoporotic Conditions ...

Bone Regeneration in Osteoporotic Conditions: Healing of Subcritical-Size Calvarial Defects in the Ovariectomized Rat Sara F. O. Durão, DDS1/Pedro S. Gomes, DDS, MSc, PhD2/José M. Silva-Marques, DDS, PhD3/ Hélder R. M. Fonseca, MSc4/João F. C. Carvalho, DDS, PhD5/ José A. R. Duarte, MD, PhD6/Maria H. R. Fernandes, PhD7 Purpose: Osteoporosis is a pathologic condition characterized by low bone mass and changes in the microarchitecture of the bone tissue. Although compromised bone strength and increased susceptibility to fracture have been established, little is known regarding the process of bone regeneration in osteoporotic conditions. Accordingly, this study sought to evaluate the intramembranous bone regeneration process in an ovariectomized rat model following the establishment of calvarial subcritical-size defects (sCSDs). Materials and Methods: Calvarial sCSDs were established in rats that had been ovariectomized (Ovx) or sham-operated 2 months previously and left to heal, unfilled, for 6 months. Bone regeneration was assessed by radiographic, densitometric, histologic, and histometric analyses. Results: Radiologic and histologic analyses showed reduced new bone formation in calvarial sCSDs in Ovx animals in comparison to sham animals. Densitometric analysis of radiologic images and histometric analysis showed significant quantitative differences between groups that converged to substantiate reduced bone regeneration in Ovx animals. Conclusions: The intramembranous ossification process is impaired in the Ovx rat model. This may suggest an impairment of the bone regeneration process in clinical conditions of postmenopausal osteoporosis and highlight the requirement for selective bone regenerative strategies in affected patients. Int J Oral Maxillofac Implants 2012;27:1400–1408 Key words: animal models, bone regeneration, osteoporosis

O

steoporosis is a prevalent disease and a major health problem, with high rates of mortality and morbidity worldwide.1 This pathologic condition is

1Lecturer,

Faculdade de Medicina Dentária, Universidade do Porto, Porto, Portugal. 2 Assistant Professor, Faculdade de Medicina Dentária, Universidade do Porto, Porto, Portugal; Laboratório de Farmacologia e Biocompatibilidade Celular – Faculdade de Medicina Dentária, Universidade do Porto, Porto, Portugal. 3Assistant Professor, Cooperativa de Ensino Superior Egas Moniz. Campus Universitário, Caparica, Portugal. 4Researcher, Faculdade de Desporto, Universidade do Porto, Porto, Portugal. 5Professor, Faculdade de Medicina Dentária, Universidade do Porto, Porto, Portugal. 6 Professor. Faculdade de Desporto, Universidade do Porto, Porto, Portugal. 7Professor, Faculdade de Medicina Dentária, Universidade do Porto, Porto, Portugal; Laboratório de Farmacologia e Biocompatibilidade Celular – Faculdade de Medicina Dentária, Universidade do Porto, Porto, Portugal. Correspondence to: Dr Maria Helena Fernandes, Laboratório de Farmacologia e Biocompatibilidade Celular, Faculdade de Medicina Dentária, Universidade do Porto, Rua Dr. Manuel Pereira da Silva, 4200-393 Porto, Portugal. Fax: +351-220-901-101. Email: [email protected]

characterized by low bone mass and changes in the microarchitecture of the bone tissue, which work together to compromise bone strength and increase the susceptibility to fracture.1 Global epidemiologic data report that 1 in every 3 women and 1 in every 50 men over the age of 50 years have osteoporosis.2 Moreover, an estimated 40% of women and 13% of men aged 50 years and older will sustain an osteoporotic fracture in their lifetime. Taking into account future mortality trends, these figures rise to 47% for women and 22% for men.2,3 As a health care problem, osteoporosis comprises a large percentage of health spending and is expected to escalate in this century, with projected costs of hip fractures alone reaching US$131 billion worldwide by 2050.4,5 Even though much attention has been given to preventive approaches and new pharmacologic and physical therapies, which aim to maintain a high level of bone mass, less attention has been directed to the study of the process of bone regeneration in osteoporotic conditions. The available data, despite being sparse, focus mainly on the process of fracture healing; delayed healing and impaired biomechanical strength have been observed in experimental and clinical studies.6–8 Moreover, some experimental studies report

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a data trend substantiating an impaired biomaterialmediated bone regeneration process in osteoporotic conditions9–11; nonetheless, some authors found no differences.12–14 Regardless, the fundamentals of the de novo bone formation process in osteoporotic conditions have not been adequately detailed. Animal models play a crucial role in bone-related research, especially within the assessment of the biologic and biomechanical characteristics of bone metabolism, orthopedic implant placement, and bone graft substitutes, in both physiologic and pathologic conditions.15–18 Moreover, experimental animals have also been used extensively to model human pathologic states, such as the osteoporotic condition.19,20 The ovariectomized (Ovx) rat is the most commonly used model for the study of osteoporosis pathophysiology, diagnosis, and therapy and has been validated as a clinically relevant model of human postmenopausal bone loss.21–23 Rodents have also been widely employed in the assessment of the bone regeneration process, in the presence or absence of implanted materials, in the standardized calvarial bone defect.17,24,25 The use of the calvarial model implies the selection of a specific defect size, in which the choice between the use of critical-size defects (CSDs) or subcritical-size defects (sCSDs), is crucial.24,26 The CSD is broadly described as the smallest wound established intraosseously that does not heal spontaneously during the lifetime of the animal, as a specific condition of failed osteogenesis for overcoming the threshold of the physiologic process of tissue repair.26 It has been used routinely to address the biocompatibility and osteogenic capacity of many candidate materials and tissue engineering approaches for bone regeneration.17 However, CSDs do not provide data regarding the process of “natural” bone healing, which can be adequately examined only in the appraisal of the regeneration process of unfilled sCSDs.25 Accordingly, sCSDs have been used in the evaluation of bone formation, especially within the assessment of the impact that a wide range of biologic processes, added substances, or pathologic conditions have on the intramembranous ossification process.25,27,28 Therefore, to address the bone regeneration process in an animal model representative of the human condition of osteoporosis, the present study sought to evaluate intramembranous bone regeneration in an Ovx rat model following the establishment of calvarial sCSDs.

MATERIALS AND METHODS Animals

This experimental study was performed under the authorization of Direcção Geral de Veterinária and upheld the technical standards for the protection of experi-

mental animals, according to Portuguese (decree no. 1005/92) and European (directive 2010/63) legislation. Fourteen nulliparous female Wistar rats, aged 6 weeks, were purchased from a certified vendor (Charles River Laboratories) and housed in plastic cages in a monitored environment throughout the study period. Animals were given a standard diet (4RF24 GLP, Mucedola) and water ad libitum. After a quarantine period, rats were randomly ovariectomized (Ovx; n = 7) or sham-operated (sham; n = 7). Bilateral ovariectomy was performed as described in the following section.

Ovariectomy and Sham Surgical Procedure

At 2 months of age, animals assigned to the Ovx group were anesthetized by an intraperitoneal injection of xylazine (10 mg/kg Rompun 2%, Bayer) and ketamine (90 mg/kg Imalgene 1000, Merial). The abdominal cavity was accessed by a 2-cm midline dorsal skin incision, which allowed for blunt dissection of the connective tissue between the skin and the muscular layer of abdominal wall. Following this, the muscular layers were opened halfway down the sides of the animal. The ovaries were identified surrounded by a considerable amount of fat and pulled out through the incision. Two ligatures were placed with absorbable 4–0 sutures (polyglactin 910, Vicryl Rapide, B Braun): one in the caudal end, between the ovary and the uterine horn, and the other one in the cranial end of the ovary. Following this, the ovaries were safely cut off and the uterine horns were pushed back into the abdominal cavity. Following inspection for abdominal hemorrhage, the abdominal muscle tissue was closed with absorbable 4–0 sutures. Finally, the skin was sutured. The animals were administered tramadol (10 mg/kg Tramal, Grünenthal) intraperitoneally for postoperative analgesia. Sham surgery, which sought to assess the effect of the intervention under study by neutralizing the placebo effect and reducing bias, consisted of the same surgical protocol with the exception of the placement of ligatures and the removal of ovaries and was performed in all animals of the sham group. In agreement with the experimental protocol, the animals were weighed monthly.

Establishment of Calvarial Subcritical-size Defects

At 4 months of age (2 months following Ovx or sham operation), surgical craniotomies were performed; the surgery sought to establish 3-mm-diameter sCSDs. Both the Ovx and sham animals were submitted to the craniotomy procedure. Animals were anesthetized using sevoflurane (Baxter) inhalation anesthesia (4% to 5% induction; 2% to 3% maintenance) (Fig 1a). A midline incision through the skin allowed access to the calvarial bone. The skin was then reflected bilaterally, and a midline periosteal incision allowed for the division The International Journal of Oral & Maxillofacial Implants 1401

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Fig 1 Surgical craniotomy.

a

b

c

Fig 1a Presurgical preparation. Fig 1b Exposure of the calvarial bone. Fig 1c Establishment of standardized bilateral, bicortical, parietal sCSDs. Fig 1d Closure of the surgical wound.

d

of the subcutaneous fascia and the bilateral reflection of the periosteal flaps, following blunt dissection, to expose the calvarial bone surface (Fig 1b). With a trephine bur with an external diameter of 3 mm, standardized bilateral, bicortical, midparietal defects were created (Fig 1c). The surgical wound was then closed in layers with 4–0 resorbable sutures (Fig 1d). Intraperitoneal administration of tramadol (10 mg/kg) was used for postoperative analgesia.

Animal Euthanasia and Tissue Harvesting

Six months after craniotomy, rats were anesthetized with intraperitoneal administration of xylazine (10 mg/kg) and ketamine (90 mg/kg) and euthanized by exsanguination. Blood was collected from the inferior vena cava into heparinized tubes and further processed for plasma separation, which was later used for biochemical analysis. To address the bone regeneration process, the calvarial bone was harvested for densitometric and histologic analyses; the uterus and the left tibia were also removed for weighing and densitometric evaluation, respectively.

Characterization of the Osteoporosis Animal Model

Biochemical Data. Alkaline phosphatase (ALP) activity and plasma levels of calcium (Ca) and phosphorous (P) were determined in an autoanalyzer. Plasma estrogen levels were determined by an enzyme-linked immunosorbent assay kit (Mouse/Rat Estradiol (E2) ELISA Kit, Calbiotech), according to the manufacturer’s specifications. Radiographic Evaluation and Densitometric Analysis of Tibias. The left tibiae were fixed in 10% buffered formalin. Radiographic imaging was conducted with a RVG intraoral sensor (Kodak RVG 5100)

and the images were processed with software (Kodak Dental Imaging Software 6.8.6.0). With respect to the x-ray beam, a conventional x-ray tube (Trophy, type 708, long cone) was used at 8 mA and 70 kV. The relative position of the sensor and the exposure time were kept constant (film-focus distance of 20 cm and exposure time of 0.2 seconds). Densitometric analysis was conducted with ImageJ (version 1.41o) in a specified region of interest (ROI) in the proximal metaphysis (see Fig 3a). The intensity of the signal was evaluated in unprocessed TIFF files and adequately normalized.

Evaluation of New Bone Formation

Radiographic and Densitometric Evaluations. The calvarial bone was fixed in 10% buffered formalin. Radiographic imaging and computer image analysis were conducted as previously described. Densitometric and regenerated area analyses were conducted with ImageJ (version 1.41o). A circular ROI, 3 mm in diameter (representing the original defect), was defined for the assessment of newly formed bone.

Histologic Processing and Analysis

Following radiographic evaluation, the calvaria samples were processed for undecalcified histologic preparation. They were dehydrated and embedded in methylmethacrylate resin before sectioning and grinding to the appropriate thickness. Sections were then stained with toluidine blue. Because of the small diameter of the defects and the requirements of the undecalcified technique, rather than using surgically created marks that aimed to identify the center line of the original defect during laboratory processing, as reported by some authors,29,30 the authors identified the most central portion of each


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Fig 2a Weight of the animals throughout the study period. *Significantly different versus sham group.

500 Animal weight (g)

Figs 2b and 2c Macroscopic images of representative uteri from sham (b) and Ovx animals (c) at 10 months of age, 6 months after surgery (bar = 1 cm).

600

400

*

*

*

*

*

*

*

*

Sham

300 200

b

100 0

Sham Ovx 2

3

4

5 6 7 8 Animal age (m)

9

Ovx

10

a

osteotomy defect and selected the section displaying the widest extent. This was the area subjected to histologic and histometric analyses, according to the methodology followed by Pryor et al.27 The sections were viewed and evaluated for new bone formation by two calibrated examiners using a binocular microscope (Nikon SMX800) at low magnifications and a light microscope (Olympus CX31 with a DP-25 digital camera) at high magnifications. Histometric analyses, according to the methodology employed by Pryor et al,27 were performed with Image-Pro Plus software (version 6.0.0.260, Media Cybernetics). The following parameters were evaluated: defect width (the distance between the margins of the original defect), bone fill (the length of newly formed bone tissue along an axis bridging the gap between the defect margins), and percentage of bone fill (calculated as the percentage of the ratio between bone fill and defect width parameters).

Statistical Analysis

Hypotheses on the distribution of continuous variables between groups were tested using the Student t test or a nonparametric test (Mann-Whitney), as appropriate. Normality distribution of variables was tested by the Shapiro-Wilk test. A significance level of 5% was considered (P < .05).

RESULTS Effects of Ovariectomy

Animals in the sham-operated group reported a slow but steady increase in body weight during the observation period. Following ovariectomy, Ovx rats

c

Table 1 Plasma Levels of ALP, Ca, P, and Estradiol in Sham and Ovx Animals at 10 Months of Age Sham ALP levels (U/L) Ca levels (mg/dL) P levels (mg/dL) Estradiol levels (pg/mL)

Ovx

Mean

SD

97.81

6.96

101.97

7.14

9.89

0.34

10.13

0.27

6.13

0.26

6.25

0.24

0.836

ND

18.15

Mean

SD

–

SD = standard deviation; ND = not detectable.

weighed significantly more than sham rats at the assayed time points (Fig 2a). The uteri removed from Ovx animals at 10 months of age displayed severe atrophy (Figs 2b and 2c) and weighed significantly less (0.084 ± 0.063 g; P < .05) than those from the sham animals (0.574 ± 0.121 g). At 10 months of age, plasma levels of ALP activity, Ca, P, and estradiol were assessed in both groups of animals (Table 1). Estradiol levels were too low to be detected in Ovx animals and were around 18 pg/mL in sham animals. No significant differences were found between the groups regarding ALP, Ca, and P levels. Radiographic evaluation revealed structural differences between groups in the trabecular architecture of the tibiae (Fig 3a). The Ovx animals displayed decreased trabecular content, which was especially noticeable at the proximal metaphysis and was confirmed by the densitometric analysis performed within the delimited ROI (Fig 3b). The International Journal of Oral & Maxillofacial Implants 1403

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Fig 3a Representative radiographic images of left tibiae of Sham and Ovx animals at 10 months of age. Fig 3b Densitometric analysis of the signal intensity within the ROI delimited in the proximal metaphysis in Fig 3a. AU = arbitrary units. *Significantly different versus the sham group.

Sham

Ovx

Signal intensity (AU)

175,000 150,000 125,000 100,000

*

75,000 50,000 Sham

a

Bone Regeneration

Radiographic images of the surgically created defect, at baseline and after 6 months of healing, are shown in Fig 4a. Image analysis allowed the observation of a centripetal formation of new bone tissue starting from the original margin of the defect in both sham and Ovx animals. In the sham group, the new bone formation process seemed to be in a more advanced stage and closer to filling the original defect compared to the Ovx group. Densitometric analysis of the reported radiographic images revealed significant differences between the two groups, with an increased intensity of signal in sham animals (Fig 4b). Moreover, assessment of the regenerated area within the defect revealed an increased value for sham animals (Fig 4c). Histologic analysis showed the presence of osteogenic activity along the margins of the defect, with new bone forming by an intramembranous process in both sham and Ovx animals (Fig 5). The typical formation of a cone with the vertex oriented toward the center of the defect could be seen, in which centripetal tissue growth was evident. In the sham animals, the regenerative process seemed to be in a more advanced stage, with more newly formed mineralized bone tissue and less fibrous tissue bridging the two margins of the defect. In the Ovx group, nonetheless, new bone formation was verified, but a larger area between the margins of the defect was occupied by fibrous tissue.

Ovx

b

Histometric analysis showed a higher percentage of bone fill in sham animals (67.69% ± 8.247%) compared to Ovx animals (50.24% ± 9.766%). High-magnification images revealed the formation of well-organized regenerating trabeculae of woven and lamellar bone within the area of newly formed bone (Fig 6).

DISCUSSION It is widely believed that osteoporosis is associated with a compromised regenerative capacity of bone, which may account for the clinical impairment seen with bone-regenerative approaches in osteoporotic models. This general belief is supported by clinical retrospective studies, which present weak evidence and do not provide any explanation or insights about the potential underlying mechanisms.31–34 Accordingly, the assessment of bone regeneration in osteoporotic conditions is of particular clinical relevance. In this study, sCSDs (3 mm in diameter) were established on the calvaria of 4-month-old Ovx and shamoperated animals (2 months after ovariectomy or sham surgery) and left to heal, unfilled, for 6 months. Assessment of bone regeneration was performed via radiographic, histologic, and histometric analyses. These surgically created defects have been used to evaluate the bone regeneration process following the establish-


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Fig 4a Representative radiographic images of the sSCDs in sham and Ovx animals (left) at baseline and (right) after 6 months of healing (bar = 1 mm).

Sham

Sham

Ovx

Ovx

Fig 4b Densitometric analysis of the signal intensity within the ROI delimited in Fig 4a at 6 months. AU = arbitrary units. Fig 4c Analysis of the regenerated area at the ROI delimited in Fig 4a at 6 months. *Significantly different versus the sham group.

1,300

80

1,200

* 40 20

Signal intensity (AU)

Regenerated area (% of total)

100

60

1,100 *

1,000 900 800 700

0 b

After 6 mo

Baseline

a

Sham

Ovx

ment of pathologic conditions or to assess the efficacy of various bone therapeutic approaches.25 In this context, sCSDs have been effective to report a decreased bone regeneration capability in rodent models of diabetes,35,36 but no reports have been published regarding its use for bone regeneration in osteoporotic conditions. The validity of the model of osteoporosis used in the present study was confirmed by the biochemical and structural changes that were verified following ovariectomy. This was established by the failure to detect ovarian tissue at the necropsy and the observation of atrophic uteri in the Ovx group, which showed significant weight differences versus the uteri of shamoperated animals. Moreover, Ovx animals showed a

Sham

Ovx

c

Sham

Ovx

Fig 5 Low-magnification microphotographs of sham and Ovx calvarial sCSDs after 6 months of healing (toluidine blue; bar = 1 mm).

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a

b

c

d

Fig 6 Photomicrographs of (a and c) sham and (b and d) Ovx calvarial sCSDs defects following 6 months of healing (toluidine blue; bars: top row, 500 µm, bottom row: 200 µm).

significant increase in body weight throughout the study period, when compared to the sham animals. This is in accordance with literature reports substantiating increased hyperphagia and augmented body weight associated with the ovariectomy-mediated disruption of the normal hypothalamic-pituitarygonadal axis cycling in adult female rats.37 Substantiating the efficacy of the ovarian ablation, plasma levels of estradiol were shown to be undetectable (by the test used) in the animals in the Ovx group. Skeletal alterations were also observed in animals submitted to ovariectomy. Radiographic analysis of the left tibiae revealed a decreased trabecular structure, especially in the proximal metaphysis. In addition to the established qualitative differences, quantitative assessment of the signal intensity by densitometric analysis showed significantly lower values in the Ovx group. This methodology has been shown to be a valid technique to determine bone mineral density, reflecting the status of the crystalline component of bone.38 The attained tibial differences between Ovx and sham animals are in accordance with literature reports. Female rat ovariectomy has been shown to cause deterioration of the

three-dimensional trabecular microstructure, notably the structure model index and connectivity density, as assessed by microtomography of the proximal tibia.39 Accordingly, a decrease in the trabecular bone mineral density of the tibia and associated morphologic changes in the Ovx rat have been reported by several authors, substantiating similarities between this animal model and the human condition of postmenopausal osteoporosis.40–42 Regarding the analysis of bone regeneration in calvaria sCSDs, conventional radiographic bone density, regenerated area assessment, and histologic and histometric analyses were used to address the intramembranous ossification process within the limits of the surgically created defects. Within the evaluated radiographic images, densitometric analysis and regenerated area assessment showed significant differences, namely a decreased amount of newly formed mineralized tissue in Ovx animals compared to sham animals. Accordingly, histologic analysis revealed reduced formation of new bone tissue extending centripetally from the margin of the defect into the center of the ROI in the Ovx animals compared to the sham animals.


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These observations were confirmed by histometric analysis regarding the percentage of bone fill. In the evaluated model, ovariectomy-induced osteoporosis seemed to impair the bone regeneration process in calvarial sCSDs. To the best of the authors’ knowledge, the bone regeneration process per se, in osteoporotic conditions, has not been addressed previously in calvarial defects. Literature reports have typically focused on the evaluation of osteoporotic bone regeneration in the presence of biomaterials; nonetheless, conflicting results have been reported. Some authors substantiated an impaired process mediated by the osteoporotic condition,9–11 while other studies observed no differences in biomaterial-mediated bone formation in osteoporotic and control animals.12–14 The differences in these results may be mostly likely related to the diversity of the used biomaterials—eg, ceramics, bioactive glasses, composites, and xenografts—as well as to the different experimental models. In either case, explanation or insights about the potential underlying mechanisms are broadly lacking. In this study, an evident reduction in new bone formation was established, and since no biomaterial was added to the regenerative milieu, the regenerative capabilities ought to be settled on the osteogenic potential of recruited precursor cells and/or the remodeling process. The possibility of impaired bone formation in osteoporotic conditions has been addressed in a few studies. The evaluation of osteoporotic bone marrow–derived mesenchymal stem cell cultures revealed similar cellular size and morphology, as well as expression of similar cell surface antigens, compared to controls.43 Nonetheless, osteoporotic-derived cell cultures differed: they had a lower growth rate and exhibited a deficient ability to differentiate into the osteogenic linage, as evidenced by decreased ALP activity, type 1 collagen synthesis, and calcium phosphate deposition.43,44 Moreover, osteoporosis-associated estrogen deficiency seems to suppress the survival of osteocytes and impair the physiologic response of osteoblasts to mechanical stimuli, detection of microdamage, and repair of aged bone.45,46 Of additional relevance, a clinical report showed, through a histomorphometric analysis of iliac crest bone biopsy specimens, an inverse correlation between the cancellous apposition rate and the osteoid volume, with an increase in the proportion of adipose tissue present in the osteoporotic bone.47 The cancellous apposition rate reflects osteoblastic activity, indicating that the increased volume of adipose tissue in the osteoporotic bone may be associated with the reduced bone formation. In accordance, osteoporoticderived mesenchymal stem cell cultures have been shown to express an increased adipogenic potential compared to control cultures.48 Overall, the data seem to support the postulated impairment of the bone formation process in osteoporotic conditions.

Additionally, excessive osteoclastic activity, which seems to lead to an imbalance in the bone remodeling process that favors bone resorption in osteoporosis, may also contribute to the decreased bone regeneration.49,50 Estrogen deficiency has been shown to enhance the production of pro-osteoclastogenetic cytokines (eg, tumor necrosis factor-alpha and receptor activator of nuclear factor kappa-B ligand) and increase the number of circulating osteoclastic precursors.51 The increased production of these cytokines may also substantiate the increased spontaneous osteoclastogenesis verified in women affected by postmenopausal osteoporosis.52 Moreover, osteoporotic conditions have been associated with an increased life span of mature osteoclasts.45 Taken as a whole, the data seem to support an excessive bone resorption process in osteoporotic conditions, which may contribute to a disturbed equilibrium between bone formation and bone resorption.

CONCLUSIONS Both impaired bone formation and disruption of the bone remodeling equilibrium seemed to converge to limit the bone regeneration process in this rat model of the human postmenopausal osteoporotic condition. The impairment of the intramembranous ossification process during the healing of calvarial subcritical-size defects in the reported model may suggest that selective bone regenerative strategies are required in osteoporotic patients.

Acknowledgments The authors reported no conflicts of interest related to this study.

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