Simultaneous Treatment of Photobiomodulation and Demineralized Bone Matrix With Adipose-Derived Stem Cells Improve Bone Healing in an osteoporotic bone defect

Abstract

Introduction: The ability of simultaneous treatment of critical-sized femoral defects (CSFDs) with photobiomodulation (PBM) and demineralized bone matrix (DBM) with or without seeded adipose-derived stem cells (ASCs) to induce bone reconstruction in ovariectomized induced osteoporotic (OVX) rats was investigated.

Methods: The OVX rats with CSFD were arbitrarily separated into 6 groups: control, scaffold (S, DBM), S + PBM, S + alendronate (ALN), S + ASCs, and S + PBM + ASCs. Each group was assessed by cone beam computed tomography (CBCT) and histological examinations.

Results: In the fourth week, CBCT and histological analyses revealed that the largest volume of new bone formed in the S + PBM and S + PBM + ASC groups. The S + PBM treatment relative to the S and S + ALN treatments remarkably reduced the CSFD (Mann-Whitney test, P = 0.009 and P = 0.01). Furthermore, S + PBM + ASCs treatment compared to the S and S + ALN treatments significantly decreased CSFD (Mann Whitney test, P = 0.01). In the eighth week, CBCT analysis showed that extremely enhanced bone regeneration occurred in the CSFD of the S + PBM group. Moreover, the CSFD in the S + PBM group was substantially smaller than S, S + ALN and S + ASCs groups (Mann Whitney test, P = 0.01, P = 0.02 and P = 0.009). Histological observations showed more new bone formation in the treated CSFD of S + PBM + ASCs and S + PBM groups.

Conclusion: The PBM plus DBM with or without ASCs significantly enhanced bone healing in the CSFD in OVX rats compared to control, DBM alone, and ALN plus DBM groups. The PBM plus DBM with or without ASCs significantly decreased the CSFD area compared to either the solo DBM or ALN plus DBM treatments.

Introduction

Osteoporosis (OP), as a global skeletal disorder,¹ is characterized by the alteration of bone microstructure, reduction of bone density, robust risk of fractures, and death. OP, with 30 to 50% global privilege in postmenopausal women, is annually diagnosed in more than 9 million females in the United States.² Animal OP models are acceptable tools for the study of different surgical treatments.³ The ovariectomy-induced osteoporosis (OVX) rat model imitates the bone deterioration caused by estrogen deficiency and demonstrates the clinical signs of postmenopausal osteoporosis (PMOP).⁴ For this reason, the OVX rat model was commonly used in PMOP research.⁵

Anabolic and antiresorptive agents are approved by the Food and Drug Administration (FDA) for the prevention and treatment of OP. These medications can be associated with breast and endometrial cancers, menstruation and thromboembolic incidents⁶. Most of the prescribed medications for OP, such as bisphosphonates (BPs), are antiresorptive. Among the different types of BPs, alendronate (ALN) is frequently chosen.⁷ Antiresorptive drugs, by reducing the accessible osteoclasts, diminish bone remodeling and the risk of fracture. However, bone fragility would not be eradicated by these medications. Since microstructural degradation cannot be compensated, fragile unremodeled bones with a high degree of mineralization remain susceptible to fracture.⁸ Moreover, BP therapy is accompanied by some gastrointestinal troubles.⁹ Major problems are involved in the healing of the broken bone by OP.⁶

The bone graft or replacement is a necessary treatment strategy in a broad spectrum of patients with bone defects. Although the autologous bone graft is known as a gold standard treatment, additional pain at the donor site and a paucity of donor tissues have limited its applicability in patients. In this context, bone tissue engineering (BTE) is being considered as a good alternative.¹⁰ BTE incorporates biomaterials, cells, and signaling factors to reconstruct bone defects. The reconstruction process is as follows: (a) cell harvest and scaffold construction. (b) Seeding the cells into the scaffold creates a “cell/scaffold” structure. Following (c) structure implantation, (d) generation of new bone will increase and scaffold will degenerate, finally, (e) bone reconstruction will be completed.¹¹

Bone scaffolds not only provide a 3D microstructure for tissue regeneration, differentiation, nutrition, and metabolism but also supply a boosted mechanical strength.¹² Demineralized bone matrix (DBM) is a bone graft extender produced from human corpse bone.¹³ It is a decalcified product that uses an acidic solution to remove mineral components.¹⁴ Spongy structure and flexibility (in the hydrated state) are essential characteristics of DBM scaffolds consisting of collagen (mostly type I and a few types IV and X), non-collagenous proteins, some osteoinductive growth factors (e.g. bone morphogenic proteins, BMP), and varying percentages of calcium phosphate.¹⁵ After decalcification, BMP could be discharged from the around mineral elements and fully exert its osteoinductive capacity. The remaining DBM collagen proteins may supply a 3D structure for the ingrowth of host capillaries, perivascular tissue, and osteoprogenitor cells into the graft.¹⁴ Increasing data have shown that DBM scaffolds can increase new bone formation and enhance in vivo healing of clinical bone defects.^16,17 Though DBM scaffolds have osteoinductive and osteoconductive properties, their osteoinductivity is often reduced by manufacturing processes, such as chemical decellularization and demineralization.¹⁸ Some studies have shown that DBM scaffold possesses only a low osteoinductive capacity.^19,20 A hybrid allograft of the seeded bone matrix with mesenchymal stem cells (MSCs) may be suggested as a method for enhancing osteoinductivity by releasing growth factors.²¹ Schubert et al showed that osteogenic-differentiated AMSCs are the best candidate to improve the angio-/osteogenicity of decellularized bone allografts.²¹

The usage of MSCs is an important element in BTE. In addition to the usage of bone marrow-derived mesenchymal stem cells (BMMSCs), adipose-derived stem cells (ASCs) have recently been targeted in the BTE because of a high proliferation rate, strong osteogenic potential, and noninvasive sampling methods.²² ASCs, similar to BMMSCs, can improve angiogenesis.²³ Several experiments have shown the beneficial potential of ASCs to promote bone repair in the various animal models of femoral and skull critical-sized defects.^24-26 Although ASCs are reviewed as a successful bone healing strategy,²⁷ there are some issues that should be addressed. The low viability of cells after implantation has attracted substantial attention. Hence, enhancing the survival of transplanted ASCs is crucial for increasing the success rate of ASC-based treatment.²⁸ The interplay between cells and scaffold structure can facilitate restorative osteogenesis by increasing the viability of stem cells and guiding the differentiation of stem cells toward the bone matrix formation.²²

Photobiomodulation (PBM), as a treatment strategy, is expected to resolve the weak potential of transplantation and viability of ASC.²⁹ MSCs and progenitor cells are particularly susceptible to PBM.³⁰ It has been shown that PBM potentially promotes the proliferation, differentiation, and growth factor secretion of MSCs through altering gene expression and activation.³¹ PBM is effective in various clinical fields such as pain attenuation, wound healing, bone healing and bone remodeling.³² Researches have also indicated that PBM impacts osteogenesis and bone regeneration in comparable animal studies.³³ PBM hastens bone healing and remodeling mainly by enhancing the deposition of hydroxyapatite and collagen in the osteoid matrix, the mechanical qualities of the bone, vascular proliferation, gene expression and protein production in the bone.³⁴

Hybrid hASCs-hDBM osteoconductive scaffolds in bone regeneration have been successfully applied in previous experiments.^22,35-37 Moreover, Schubert et al showed that osteogenic-differentiated ASCs are the best candidates to improve the angio-/osteogenicity of decellularized bone allografts.³⁸ This research aimed to test the impact of simultaneous treatment of PBM and hybrid BTE scaffolds (including human[h] DBM, and hASC) on accelerating the healing rate of a critical-sized femoral defect (CSFD) in an OVX rat model.

Materials and Methods

Animals and Study Design

Seventy-two female Wister rats (3 months old, 200–250 g) were used in this research. The animals were caged under environment-controlled conditions (12-hour light/dark cycle, 22-25°C). A standard laboratory diet (Behparvar, Tehran, Iran) and tap water were available ad libitum. The animals were anesthetized by the intraperitoneal administration of xylazine and ketamine and then subjected to OVX.⁴⁰ Bilateral CSFD surgeries were done 14 weeks later.

The rats were randomly divided into six groups (each group, n=6 rats). (1) In the control group (C), CSFDs were not treated. DBM was transplanted into the CSFD in groups 2-6 (DBM-CSFD). (2) S group, (DBM-CSFD). (3) S + PBM group, the DBM-CSFDs were treated with PBM. (4) S + ALN group, the DBM-CSFDs were treated with 0.01 mg/kg/d ALN subcutaneously.³⁹ (5) S + ASCs group, ASCs were seeded into DBM and then implanted into the CSFD. And (6) S + PBM + ASCs group.⁴¹ At the fourth (anabolic phase of bone healing) and eighth week (catabolic phase of bone healing) post-operation, six rats from each group were euthanized and the left and right distal femurs were isolated and CSFDs were examined by the cone beam computed tomography (CBCT) scan and histology respectively. Groups 2-6 were considered as treatment groups.

Procedure for Ovariectomy

Bilateral OVX was conducted on female rats according to a previously described method. In brief, after exposure of abdominal cavity, 2 ovaries were exposed and excised from the rats under sterile condition and general anesthesia. The rats were housed for 14 weeks in order to establish OP. Ceftriaxone, as the antibiotic therapy, was administered.⁴⁰

Preparation of the Demineralized Bone Matrix Scaffold

The DBM was a donation from Hamanand Saz Baft Kish Company (TRC Corporation, Kish, Iran). It was prepared from the cancellous sections taken from the diaphysis of the long bones of human cadavers under sterile conditions. After cutting the bones into the small pieces, a series of physical and chemical procedures were performed to remove mineral content, blood, fat and cells.⁴¹

Isolation, Expansion, and Immunophenotyping of Human ASCs

hASCs were purchased from Royan cell bank institute (code: RSCB:0591). The flow cytometry analysis was performed to characterize the hASCs based on previously reported MSC markers.^41,42

Seeding hASCs on DBM Scaffolds

The DBM scaffolds were cut into cubes (3 mm in diameter to 5 mm in thickness) and transferred into 24‐well plates. Each DBM scaffold was incubated with 10 µL of enriched hASCs (passage 4, 3×10⁴/µL density) for 2 hours. Then 150 µL of the culture medium was added to each well. The wells were incubated at 37°C and 5% CO2 for 24 hours to allow cell adhesion. Finally, each cube of the DBM (containing hASCs) was implanted into the CSFD of the rats (groups 5, and 6).⁴¹

CSFD Model and DBM Scaffold Transplantation

Fourteen weeks after the OVX surgery, the rats were anesthetized by intraperitoneal injections of ketamine (50 mg/kg,) and xylazine (5 mg/kg,).⁴³ Standardized 3 mm femoral cylindrical defects were drilled in the distal metaphysis of the femurs of both sides (2 defects in each rat). The defects in groups 2-4 were filled with the DBM scaffold. In the S + ASCs and S + PBM + ASCs groups, the bone defect was filled with the hybrid hASCs -DBM scaffold.⁴¹

Photobiomodulation

CSFD in groups 3 and 6 were immediately subjected to PBM (MUSTANG 2000, LO7 probe; Technica Co, Moscow, Russia) after surgery. PBM therapy continued 3 days a week for 4 and 8 weeks on the basis of the time of the euthanization.⁴¹ The rats were sedated with half dosages of anesthetizing drugs which were used for general anesthesia.⁴³ PBM was performed in each session at three points; one point was exactly over the CSFD, the second point was 1 cm proximal to the center of the CSFD, and the third point was 1 cm distal to the center of the CSFD. The PBM parameters are shown in Table 1.^41,44

Table 1. Photobiomodulation parameters in the study.

Parameters	Dose and unit
Peak power output	75w
Average power	0.001
Wavelength	890nm
Pulsed frequency	80Hz
Spot size	1cm2
Pulsed duration	180ns
Duration of exposure of each point	900 seconds
Energy density for 1 shooting, 1session(1day), 1week, 4weeks, and 8weeks	0.972(J/cm2), 2.96(J/cm2), 9.74(J/cm2), 34.99(J/cm2), 69.98(J/cm2).

CBCT Imaging and Measurement of the Bone Defect Area

Four and 8 weeks after implantation, the rats were sacrificed. The removed femora were fixed in 10% formalin saline for 48 hours and subjected to the CBCT imaging system (PaX-i3D Green of Vatech scanner, Gyeonggi-do, Korea). During bone defect scanning, 50 kVp, 2 mA, 0.3 mm voxel size and 50 × 50 mm field of view were used. The scanned slices were reconstructed into DICOM files. The bone defect area of CSFDs in the studied groups was measured. The bone defect area (%) was calculated according to the residual defect area on a given week (tx) relative to the measured defect area on the day of surgery (3 mm) as follows:

Histological Examination

After CBCT scanning, the femurs were decalcified in 10% EDTA for two weeks, and the EDTA solution was changed twice a week. Sections with 5 μm thicknesses were cut with a microtome and stained with the hematoxylin and eosin (H&E) method. New bone formation at the bone defects was observed and examined descriptively by an optical light microscope (Olympus, Japan).⁴⁵

Prior to cell transplantation, the hASCs were marked with 1,1’- dioctadecyl-3,3,3’,3’- tetramethyl indocarbocyanine perchlorate (DiI, Invitrogen)46. The hASCs were ready by a standard technique. Initially, 50 µg of DiI was added to 50 mL of DMSO. Then, 1 × 106 cells were exposed to 5 µL of DiI per mL of the medium at 37 °C for 30 min and 4 °C for 15 minutes. Next, the cell suspension was centrifuged, the media was eliminated, and the cells were twice washed in sterile PBS. Subsequently, DiI-marked hASCs were seeded into the DBM scaffolds and checked to utilize a fluorescent magnifying lens (Olympus, Tokyo, Japan). At four and eight weeks after transplantation of the marked hASCs, the CSFDs were removed, and histological sections of the CSFD were ready and stained with the H&E strategy. The color intensity of the DiI-positive region in the fracture bed of S + ASC and S + PBM + ASC groups was calculated utilizing the Image J program.⁴⁶

Statistical Analysis

The data are presented as mean ± standard deviation (SD). The normal distribution of the data was analyzed by the Shapiro test. One-way analysis of variance (ANOVA) with the least significant difference (LSD) post-hoc test was applied. A P value of <0.05 was considered statistically significant. The Mann-Whitney U test was used for statistical analysis of non-parametric data.

Results

hASCs Stem Cell Surface Marker Characterization

One week after the initial isolation of hASCs, the flasks were filled with cells. Spindle-shaped adherent cells were observed at passage 4 (Figure 1A). To examine the cell phenotype, passage 4 of the hASCs was characterized. Characterization of cell surface marker by flow cytometry indicated that a high percentage of hASCs (Figure 1B–E) were positive for CD105 (84.8%) and CD73 (99.8%) as markers of MSCs. The percentages of positive cells for hematopoietic markers including CD45 (0.4%) and CD31 (0.3%) were low. The evaluated surface markers indicated the true stemness of hASCs population.

Figure 1.

Identification of hASCs. (A). hASCs at Passage-4 sat 100×magnification under the inverted microscope (B). hASCs were positive for CD105 (C) and CD73 (D) as MSCs markers. Low percentages of cells were positive for CD45 (D) and CD31 (E) as hematopoietic markers.

DiI Labeling

Figure 2 displays the magnified micrographs of sagittal sections of stained H&E specimens obtained from the S + ASCs and S + PBM + ASCs groups 4 and 8 weeks post-implantation. In the histological sections, numerous DiI-positive (red) cells were identified throughout the defect (Figure 2A-D). The results indicated that the implanted hASCs survived throughout the experimental time and migrated to the defect.

Figure 2.

Imaging of hASCs Migration in CSFD. Sections of paraffin-embedded tissue were prepared at 4 and 8 weeks following the transplantation of hASCs labeled with DiI dye (10×magnification. Scale bar, 300μm). Panels A and B show DiI positive areas in the S + ASCs group. Panels C and D show DiI positive area in group S + PBM + ASCs.

Outcomes of CBCT Evaluation

The CBCT imaging at the fourth and eighth weeks after bone defect induction revealed that the bone defect mostly remained empty in the control group. It also illustrated poor bone healing and shattered bone structure near the implantation site in the S, S + ALN and S + ASCs groups (Figure 3A). It seems that applied treatment strategies in these groups failed to heal the defects. In contrast, the S + PBM + ASCs group triggered new bone formation in the fourth week after surgery. In this group, a large area of the femur recovered and only small defect lesions remained in the eighth week after surgery. The S + PBM group not only elicited abundant new bone formation in the fourth week after surgery, but also almost filled the defect in the eighth week after surgery. As is shown in Figure 3b, the three-dimensional CT images of CSFD showed that the greatest amount of new bone formation occurred in the S + PBM and S + PBM + ASCs groups in the fourth and eighth weeks after surgery. The S + PBM group regenerated the highest amounts of mineralized bone in the eighth week after surgery.

Figure 3.

Bone Healing as Evaluated by CBCT. A: CBCT Images of the defect area in the 4th and 8th weeks. B: frontal view of reconstructed three-dimensional CT images of rat femurs in different groups. The open arrowheads indicate the defect regions. n = 5 in each group.

Figure 4 shows the percent of the defect area. At the fourth week post-surgery, the treatment groups (2-6) compared to the control group showed a significant decrease in the defect area (Mann-Whitney test, all P = 0.008). S + PBM treatment significantly decreased the defect area compared with the S and S + ALN treatments (Mann-Whitney test, P = 0.009 and P = 0.01). S + PBM + ASCs treatment significantly decreased the defect area compared with the S and S + ALN treatments (Mann-Whitney test, P = 0.01) (Figure 4, Panel A).

Figure 4.

A-B: Comparison of the defect area percentages in the experimental groups. C: Measurement of the defect area as assessed by CBCT. Mann-Whitney test; * P < 0.05 and ** P < 0.01.

At the eighth week post-surgery, the control group showed a significant increase in the defect area compared with the treatment groups (Mann-Whitney test, P = 0.01, P = 0.009, P = 0.03, P = 0.02 and P = 0.009, respectively). The defect area in the S + PBM treatment was significantly smaller than the S, S + ALN and S + ASCs treatments (Mann-Whitney test, P = 0.01, P = 0.02 and P = 0.009) (Figure 4B). While the defect area in the S + PBM + ASCs group reached 0.9 mm, it subsided to 0.5mm in the S + PBM group (Figure 4C).

Histological Findings

At the 4 and 8 weeks after surgery, while defects were filled with fibrous tissue in the control group, at the same time the newly formed bone was observed in the treatment groups. The extent of new bone formation in the S + PBM, S+ASCs and S + PBM + ASCs groups was greater than the control group. Compared to the other treatment groups, the defects of the S + PBM group exhibited much more new bone formation and many defects had disappeared. Moreover, the defect areas exhibited maximum bone formation (from the defect margin to the center), remodeling and consolidation in the S + PBM + ASCs group, compared to the other ones (Figure 5).

Figure 5.

Photomicrographs of CSFD From Each Group at 4 and 8 Weeks (W) After Surgery; H/E staining; original magnification, X4. The image of the control group shows that most parts of the hole persisted in the fourth and eighth weeks after surgery. In both S + PBM and S + PBM + ASCs groups, the hole was almost restored by the new bone formation in the eighth week after transplantation. FT, fibrous tissue; NB, new bone; S, scaffold; PBM, photobiomodulation; ASCs, adipose-derived stem cells.

Discussion

The design of new innovations and instruments, by providing patient comfort and improving care outcomes in less time, has made the medical procedures easier to be carried out.⁴⁷ Our present results demonstrate the positive impacts of the combination of PBM and DBM with or without ASCs on inducing bone regeneration and improving bone repair.

Autogenous bone grafting is known as the gold standard for healing bone fractures. Meanwhile, certain drawbacks such as donor site morbidity and insufficient bone resources cannot be resolved.⁴⁸ BTE techniques, including osteogenic cells, scaffolds, and either external or in situ biostimulators (signaling) such as PBM, are very encouraging as a solution for the management of non-healing large bone defects. Hence, they have recently been introduced as an alternative to conventional treatments. BTE techniques show huge potential for stimulating bone repair and overwhelming some of the disadvantages of bone grafting.⁴⁹ Seebach et al compared various biological and synthetic scaffolds for choosing a suitable one in bone replacement therapy. Intriguingly, bone allografts relative to all other scaffolds displayed the highest cell conductivity probably due to the collagen-fiber structure.⁵⁰ DBM has long been applied for bone transplantation. BMPs and other growth factors in the DBM make it capable to induce osteogenesis. During bone repair, BMPs play an essential role in the development and differentiation of several types of cells. They recruit the MSCs to the healing site and stimulate their alteration to a bone-forming lineage.³⁵ The osteoinductivity of DBM relies on the BMP-2 and BMP-7 levels as the key growth factors. Variability in the osteoinductive potential of human DBM products is due to different extraction and processing procedures of DBM that can reduce the level of BMP in the sterilization and storage procedures. Whereas the product is subject to the same procedure, the difference depends on the quality of allogeneic bone donors. In terms of therapeutic application and effectiveness, although DBM has some prospects, confirmative evidence to support its singular use as a bone substitute is restricted.⁵¹ Hence, PBM and BTE offer an opportunity to improve the osseointegration of DBM. We aimed to increase the osteogenic properties of DBM by BTE and PBM approaches to treat femoral bone defects.

Postmenopausal women with OP have a high risk of fracture. Several drugs have been used for the treatment and prevention of OP and fractures. The side effects of medications to treat OP tend to reduce the life quality in affected persons.⁵² BT is the gold standard for restricting bone loss. The bone loss that occurs in numerous conditions such as age-related OP in both males and females is characterized by increased osteoclast-mediated bone absorption.⁵³ Although radiographic and histological data revealed that the application of ALN plus DBM treatment improved bone healing relative to the control group, no differences were observed between the ALN plus DBM treatment and other therapeutic groups. These conflicting data could be explained by the fact that long-term BT might actually inhibit bone remodeling to such an extent that it disturbs the normal healing of the bone and increases the risk of fracture.⁵⁴ A research by Kennel et al showed that negative impacts of BT (such as ALN) might restrict BT prescription for OP in some patients and best usage of BT for OP needs suitable calcium and vitamin D intake.⁵⁵ Accordingly, the lengthy treatment of OP cases with nitrogen-including BT has been linked with uncommon fractures in the femur.⁵⁶ ALN, which is often prescribed for OP, has been linked with various adverse effects in different organs of the human body, such as gastrointestinal, endocrine, and musculoskeletal systems.⁵⁷

MSCs have been demonstrated to be the most appropriate stem cells for bone regeneration. In addition to BMMSCs, the applicability of ASCs in the BTE has been rising in the previous years.²² It is worth noting that BMMSC sampling is quite invasive and painful, with possible morbidity and poor outputs. On the contrary, ASCs sampling raises outputs and decreases the pain in a simple procedure. Adipose tissue also contains considerably more stem cells than bone marrow (5% vs. 0.01%).⁵⁸ Various studies have documented that ASCs relative to BMMSCs may have equal or even superior regenerative capacity (1) due to a higher cell proliferation rate (2), higher regenerative factor secretion (3) and higher immune suppression capacity.⁵⁹ In different animal studies, such as rat and canine models of skull defects, femoral head osteonecrosis, femur defect, disruption osteogenesis, and spine fusion, the osteogenic ability of ASCs was demonstrated alone or in combination with a scaffold. Different scaffolds may be utilized for ASCs seeding, involving apatite-coated poly (lactic-co-glycolic acid) scaffolds, collagen-ceramic runners, type I collagen matrix and coral scaffolds.⁶⁰ To date, animal studies to repair bone defects have used scaffold transplantation, cell transplantation and hybrid scaffold-cell transplantation. In addition, the transplantation of a hybrid scaffold-cell structure has been shown to have greater osteogenic ability than that achieved by co-transplantation of scaffolds and MSCs. Important considerations in the transplantation of MSCs involve how the cells can be transferred to the surgical site or how the inserted cells can be functionally grafted onto it. It is believed that isolated MSCs cannot themselves be fixed and readily stay in the region of a bone defect. Thus, the MSCs must be added along with any carriers or scaffolds.⁶¹

Our findings revealed that the treatment of CSFD with ASCs-seeded DBM scaffolds (S+ASCs group) significantly decreased the defect area relative to the control group. This analysis revealed the significance of ASCs in bone healing. Kloeters et al, in an experiment, showed the pro-osteogenic impact of ASC-seeded bone allografts in rabbits.⁶² Schubert et al investigated that bone allografts were colonized with ASCs and BMMSCs in a murine ectopic transplantation model.²¹ Various experiments on the ASCs, including co-culture with bone allografts or synthetic scaffolds, demonstrated an increase in osteogenesis and angiogenesis potentials of hybrid scaffold.^2-4 In terms of histological analysis, the S+ASCs treatment displayed more new bone formation relative to the control group. However, The CBCT images showed that treatment with ASC-seeded DBM scaffolds failed to completely fill the defect area. This result indicated that ASC-seeded DBM scaffolds can form new bone in a small area of femoral defects. Nevertheless, the number of migratory cells might be not adequate to allow new bone to cover the CSFD completely.

Recent studies have demonstrated a substantial decline in survivable MSC numbers in animal models of skin injury and skull defects 14 days after implantation.^63,64 The main reason for unacceptable clinical outcomes of ASCs transplantation may be either the poor biological activity or the poor survival ability of injected ASCs.⁶³ There are also attempts to apply new approaches in improving the potentials of survival, homing to the inflammation site, and immunomodulation. To our knowledge, the simultaneous healing effect of PBM and hybrid ASCs-seeded DBM on patients with CSFD or animals with OP has not been reported yet.

Various experiments have indicated that PBM, by increasing the number of differentiated osteoblastic cells, enhances osteoid tissue and bone formation. It is effective through inducing osteogenic cell proliferation, modifying the activity of osteocytes and inducing the differentiation of precursor cells.³³ Accordingly, our findings showed that treatment with PBM and ASCs-seeded DBM relative to other treatments accelerated the healing rate of CSFD in rats as analyzed by the H&E. Incremental bone healing after PBM treatment was also observed in the CBCT analyses. The simultaneous treatment with PBM and hASC-seeded DBM demonstrated a statistically significant reduction of the defect area compared to either the solo DBM or ALN plus DBM treatments at the fourth week (Figure 4A). Some experiments have shown that PBM can stimulate either the mesenchymal cells indirectly or osteoblasts directly, which may lead to decreased bone mass deposition and secretion of matrix elements in the bone defects and fractures.^65,66 Choi et al declared that the use of ASCs in an acellular dermal matrix concomitant with PBM exposure can substantially enhance bone formation and drastically increase the mineral density and mass volume of the bone.⁶⁷

Interestingly, we found that the simultaneous administration of PBM and DBM hastened the bone healing of CSFD in the OP rats. Additionally, the CBCT photographs revealed that the simultaneous treatment of CSFD with PBM and DBM not only elicited abundant new bone formation in the fourth week after surgery, but also filled the defect in the eighth week after surgery (Figure 3A). It was noted that owing to its effects, PBM helps the process of new bone formation and reduces the healing time of the defects. Bosco et al demonstrated that PBM with or without the bovine bone graft could improve bone formation in rats with critical-sized calvarial defects.⁶⁷ Cunha et al found that PBM improved the repair of bone defects.⁶⁹ Fernandes et al demonstrated that Biosilicate®/PLGA composites in association with PBM exposure by increasing the rate of material dissolution can increase the amount of newly formed bone tissue at the site of defects.⁷⁰ Consistent with these previous findings, we showed that the simultaneous application of PBM and DBM resulted in an accelerated reduction in the defect area (Figure 4B-C). The increased deposition of hydroxyapatite and collagen in the osteoid matrix at the site of CSFD might be responsible for improving the healing rate of the bone. The experimental S + PBM group relative to S, S + ALN and S + ASCs groups showed smaller defect areas at both times, mainly at the eighth week (Figure 4B). This result suggests that PBM increases the osteoconductive potential of biomaterials. Considering the osteoinductive properties of biomaterial and PBM, it can be postulated as an addictive action. Other authors have reported that the use of biomaterials in laser therapy can enhance the effectiveness of light.⁶³

The present study had some limitations. First, we did not verify the optimal quantity of seeded ASCs to affect the bone repair. For example, little implanted cells might leave a dead space or cause other problems. Second, we did not verify the abilities of ASCs seeded DBM or PBM in a bone fracture model, which is relevant as more cases of bone fracture are encountered in clinical practice than of bone defects. Third, we did not demonstrate the efficacy of PBM and BTE approaches in a larger animal or a more massive bone defect model. Another limiting factor for our study was the unavailability of the micro-CT; thus, we used CBCT for evaluating the healing of bone defects.

From a clinical point of view, regeneration of 80% or more within a certain time should be considered necessary for applying a BTE as an alternative to autologous bone transfer. This remains quite a challenge, especially for large-sized defects.²² In the present study, CT scan evaluation revealed that the percentage of defect areas in the simultaneous PBM and hASC-seeded DBM treatment reached up to 32.5%, whereas treated defects with PBM and DBM decreased to 17.3% (Figure 4C). On the other hand, the percentage of healed areas in the S + PBM group at the 8th week was 82.7%. The present study demonstrated the therapeutic potential of PBM and DBM without cell seeding in the animal model of long bone defects. Accordingly we found that ASC, and PBM significantly increased healed bone strength in an experimental model of DBM-treated CSFD in the catabolic phase of bone healing in osteoporotic rats.⁴¹ However, ALN alone and PBM plus ADS were superior to the other protocols.

The present study demonstrated that the simultaneous hDBM + PBM treatment of CSFD significantly enhances the bone healing process in the OVX rats. Although several issues including biomechanical and molecular mechanisms of hDBM + PBM effectiveness in bone healing remain to be investigated, these results confirmed that the simultaneous PBM and DBM treatment with or without ASCs seeding could improve the fracture repair in vivo. It suggests the probable benefit of PBM exposure to improve the outcomes of BTE treatment in large bone defects.

Conclusion

The combination of PBM and DBM with or without ASCs significantly enhanced bone healing in the CSFD in rats compared to the control, DBM alone, and ALN plus DBM groups. The simultaneous treatment with PBM and DBM with or without ASCs significantly decreased the CSFD area compared to either the solo DBM or ALN plus DBM treatments.

Authors’ Contribution

MA researched data and wrote the first draft of the manuscript with the help of MK, RG, FZ, PK, AK, and RBA performed experiments. MAA, AA, FFF, and MN made suggestions. SC and MB edited the paper scientifically.

Ethical Considerations

The protocol of animal treatment was approved by the Animal Ethics Committee of Shahid Beheshti University (file no: IR.SBMU.MSP.REC.1397.533).

Conflict of Interests

The authors declare that they have no conflict of interest.

Acknowledgments

Demineralized bone matrix was generously donated by Hamanand Saz Baft Kish Company (TRC Corporation, Kish, Iran).

Please cite this article as follows: Gazor R, Asgari M, Abdollajhifar MA, Kiani P, Zare F, Fadaei Fathabady F, et al. Simultaneous treatment of photobiomodulation and demineralized bone matrix with adipose-derived stem cells improve bone healing in an osteoporotic bone defect. J Lasers Med Sci. 2021;12:e41. doi:10.34172/jlms.2021.41