Human umbilical cord mesenchymal stem cells accelerate and increase implant osseointegration in diabetic rats

Abstract Objective This study was conducted to assess the effect of hUCMSCs injection on the osseointegration of dental implant in diabetic rats via Runt-related Transcription Factor 2 (Runx2), Osterix (Osx), osteoblasts, and Bone Implant Contact (BIC). Methodology The research design was a true experimental design using Rattus norvegicus Wistar strain. Rattus norvegicus were injected with streptozotocin to induce experimental diabetes mellitus. The right femur was drilled and loaded with titanium implant. Approximately 1 mm from proximal and distal implant site were injected with hUCMSCs. The control group was given only gelatin solvent injection. After 2 and 4 weeks of observation, the rats were sacrificed for further examination around implant site using immunohistochemistry staining (RUNX2 and Osterix expression), hematoxylin eosin staining, and bone implant contact area. Data analysis was done using ANOVA test. Results Data indicated a significant difference in Runx2 expression (p<0.001), osteoblasts (p<0.009), BIC value (p<0.000), and Osterix expression (p<0.002). In vivo injection of hUCMSCs successfully increased Runx2, osteoblasts, and BIC value significantly, while decreased Osterix expression, indicating an acceleration of the bone maturation process. Conclusion The results proved hUCMSCs to accelerate and enhance implant osseointegration in diabetic rat models.


Introduction
Diabetes is a chronic systemic disease and one of the biggest public health problems in the world. People with uncontrolled diabetes are more prone to tooth loss because of periodontal problem, decreasing the quality of life due to disturbances in the masticatory system. Edentulous people will have unsuccessful dietary management, which is essential to maintain blood sugar levels in the body. 1,2 One way to overcome this issue is by making dentures. There are two types of dentures, removable and fixed. Currently, the first choice for fixed denture is dental implant, 3 since it has many advantages, such as preservation of dental tissue, bone, improves mastication almost like natural teeth and resistant to caries. 4 On the contrary, people with uncontrolled diabetes have poor metabolic conditions that can affect the osseointegration process of dental implants. A previous study showed that experimental diabetic rats had a decrease in Bone Implant Contact (BIC) up to 50% and did not reach normal value until the 80 th day post implant placement. Moreover, the volume of bone around the implant body also decreased by 50%. These conditions may impact the failure and prolonged dental implant osseointegration process. 5 Osseointegration is a term used to describe the integration between bone and implants. 6 The osseointegration mechanism occurs when direct contact between the bone and implant body take place.
To determine the success of osseointegration process, histological evaluation and histomorphometry can be carried out on experimental animals by examining BIC. The success rate of dental implants in patients without systemic diseases is between 90 and 95%, 10 years post-insertion. 7 However, in patients with systemic diseases such as uncontrolled diabetes, the success rate decreases especially in the first 2 to 4 weeks after implant placement. 8 Several studies have been carried out to overcome chronic diabetes complications using stem cells to improve cell regeneration. Recently, Human Umbilical Cord Mesenchymal Stem Cells (hUCMSCs) are widely used because they are multipotent, nonhematopoietic, have paracrine mechanism, can self-repair and differentiate into other cells, such as osteoblasts, adipose tissue, and chondroblasts. 9 hUCMSCs are abundant source, painless collection with no ethical restraint, and have minimal immunogenicity.
Compared with other source of stem cells such as bone marrow (BM) or peripheral blood, hUCMSCs have more advantages. hUCMSC can increase the formation of new blood capillaries so that blood circulation in ischemic areas increases. 10 Furthermore, hUCMSCs were applied to treat severe pulmonary arterial hypertension and increased activity of regeneration and anti-inflammation properties, improving clinical parameter of three year old females. 11 hUCMSCs also significantly improves IL-4, IL-6, and IL-10 expressions, reduce the cytokine storm, and modulates NK cells from severely ill COVID-19 patients. 12 The injection of hUCMSCs was examined in the osteoporotic mandible and showed significant results in increasing bone density. 13 The use of hUCMSCs in accelerating the osseointegration process of implants in diabetic patients was never done.
Several markers can determine the osseointegration process, such as Runt-Related Transcription Factor 2 (Runx2), Osterix (Osx), osteoblasts, and BIC. The aim of this study was to determine the effect of hUCMSCs on Runx2, Osx, osteoblasts, and BIC as essential markers to examine the acceleration of osseointegration process of dental implants after hUCMSCs injection in diabetic rat model.

Implant specification
The implant (Titanium grade 1) is cylindrical with 1 mm diameter and 2 mm height, machining by CAD/ CAM (Yoshimi Inc. Osaka, Japan).

Number of sample and sample group
In total, 28 Wistar rats were divided into 4 groups (7 rats each). C1 was the implant group and terminated after 2 weeks, C2 was the implant group and terminated after 4 weeks, T1 was the implant group with hUCMSCs injection and terminated after 2 weeks, while T2 was the implant group with hUCMSCs injection and terminated after 4 weeks.

Implant placement
Before anaesthesia procedures, rats were fasted for 8 hours before implant placement. Ketamine 10% 1 cc and Xylazine 1 cc were injected intramuscularly.
The osteotomy area was only located on the right femur, which was shaved and disinfected with Povidone Iodine 10% (Betadine, Indonesia). All the surgical instruments and implants were sterilized with  (4) Note : ** indicated animal died after STZ injection intraperitoneally  body weight intramuscular were administered. Figure   1 shows the diagram of the implant placement.
After receiving treatment, all animals presented limited movement for 3 days. However, no lack of appetite nor weight loss or death were observed.
After 2 and 4 weeks, the rats were euthanized using perfusion technique (1-cc Ketamine 10% and 1-cc Xylazine, intramuscular). The area of interest was cut at 0.5 mm radius from outer implant margin. The specimen was soaked into 10% buffered formalin for a week, continued with 10% ethylene diamine tetra acetic acid (EDTA) as much as 50 times volume of the specimen's volume.

Immunohistochemistry
After deparaffination and rehydrate tissue section, the specimens were washed twice in buffer (Biogear).
To reduce non-spesific background staining due to endogenous peroxidase, slides were incubated in Hydrogen Peroxide Block (Thermo Scientific, USA) for 10 minutes, then washed four times in buffer (Biogear). Ultra V Block (Thermo Scientific, USA)

Results
All animals induced with STZ intraperitoneally for five consecutive days presented hyperglycemia (> 300 mg/dL) after the fifth day (Table 10. About 10% of the rats treated with STZ (4/32) died after induction. Furthermore, randomization was carried out before implant placement and hUCMSCs injection (Table 1). Blood Glucose Level was monitored on each animal before implant placement and every week until termination day. The results showed that the target hyperglycemia for diabetic animal model was maintained > 300 mg/dL ( Table 2).  Figure 3 shows the comparison of Runx2 expression among groups. The lowest Runx2 expressions were found in the C2 group (4.51 ±1.4), while the most abundant expressions were on T1 group (9.26 ±2.13). C1 and T2 group was having similar number of expressions, 6.26 ± 2.02 and 6.43 ± 2.01, respectively. Significant difference was found between C1 and T1 group (p < 0.01), C2 and T1 group (p < 0.001), T1 and T2 group (p < 0.05). and T1, p < 0.01), C2 and T2, p < 0.01), but was found no significant difference between the control groups (C1 and C2) and between the treatment groups (T1 and T2). The lowest number of osteoblasts was found in C2, while the highest was in T2.

Runt-Related Transcription Factor 2 (RUNX2)
Bone implant contact (BIC) Figure 6 shows (BIC) among groups. The highest BIC was found in T2 group (77.29% ±14.29%), and the lowest in C1 group (29.71% ± 9.99%). The C2 and T1 group mean value of BIC length was 54% ± 15.36% and 67.29% ± 18.35%, respectively. Significant difference was found between C1 and C2 group (p < 0.01), C1 and T1 (p < 0.001), C1 and T2 (p < 0.000), and C2 and T2 (p < 0.01). No significant difference was found between treatment groups. In this study, hUCMSCs remained viable during the procedure. This result was also similar to previous study by Hendrijantini,et al. 45 (2021) (5) MSCs can also increase PGE2 secretion that drives resident macrophages with an M1 proinflammatory phenotype toward an M2 anti-inflammatory phenotype, which produce higher amounts of the anti-inflammatory cytokine IL-10 and contribute to inflammation resolution. 31,32 To determine the success of the dental implant osseointegration process, several markers that play an important role can be used, such as Runx2,Osx,osteoblasts,and BIC. 22,33 Runx2 is a transcription factor that plays a role in skeletal growth, osteoblast The results showed that there was no significant difference between groups C1, T1 and T2; however, in group C2 the number of Runx2 expressions was minimal. The absence of differences in Runx2 expression in groups T1 and T2 may indicate that the maximum number of Runx2 may have been reached before the end of two weeks. Thus, in groups T1 and T2 the amount of Runx2 expressions did not differ statistically. This is also supported by the BIC data that shows high contact between implants and bone in the treatment group. BIC percentage from the treatment group has fulfilled the minimum BIC required for implant success, which ranges from 50% to 80%. 39 However, the amount of Runx2 was still high in group C1 and decreased significantly in group C2, indicating that the maturation process is likely to occur between two and four weeks. Therefore, the Wolff's law, when an implant is placed in the jawbone, its microenvironment changes and internal structure in response to implant placement and loading. Clinically, the minimum BIC required for implant success is from 50% to 80%. 39

Discussion
The previous study in diabetic patients reports a delay in the process of bone formation and remodelling.
Studies using experimental diabetic rats terminated 2 weeks after implantation, showing an average BIC of 28.82% and 56.55% for non-diabetic rats. After 6 weeks, the studies showed an average BIC of 51% for diabetic rats and 66.4% for non-diabetic rats. In this study, local infiltration with insulin was given to the implant site with an average BIC for 2 weeks of 50.73% and 58.3% for 6 weeks, both significantly lower when compared to the non-diabetic group. 42 The use of Nerve Growth Factor to increase BIC in diabetic experimental animals showed significant results with termination times of two, four, and eight weeks. In the two week data analysis, the mean BIC was 36.97% for the non-diabetic group, 22.11% for the diabetic group, and 36.97% for the NGF group. For the four week group, BIC increased 55.46% for the non-diabetic group, 42.61% for the diabetes group, and 54.34% for the group with NGF administration.
Meanwhile, for the six week group the average BIC was 65.44% for the non-diabetic group, 55.75% for the diabetes group, and 67.99% for the group receiving NGF. 46 This study observation lasted up to 4 weeks. For future studies, we suggest adding more time interval, thus the peak level of each expression can be known and the exact mechanism can be revealed. In this study, we also did not compare the use of insulin as a control positive group. Many factors were unknown and need to be further explored. Hopefully, this study can be a start for future development of hUCMSCs as a treatment for dental implant osseointegration under diabetes mellitus circumstances.
In this study, the highest BIC was in the T2 group and the lowest was in the C1 group. The mean BIC data for the C1 and C2 (30% and 54 %) are almost similar with the data from previous studies on implants in diabetic experimental animals. 42 For stem cells treatment groups, hUCMSCs increased the mean BIC to 67% and 77%. Compared to other studies such as local insulin injection and NGF administration, hUCMSCs could potentially accelerate and increase BIC. Moreover, hUCMSCs increased the two weeks and four weeks BIC in diabetic animal higher than in non-diabetic group. Conclusion hUCMSCs successfully accelerated and increased dental implant osseointegration in diabetic condition regarding Runx2, Osterix, osteoblasts, and BIC at two and four weeks examination.