Morphological Characteristics and Correction of Long Tubular Bone Regeneration under Chronic Hyperglycemia Influence

Abstract

Introduction。Unsatisfactory consequences of bone regeneration disorders in diabetes mellitus (DM) patients, their high prevalence, complication number, and difficulties in treatment require further study and deeper understanding of reparative osteogenesis mechanisms under chronic hyperglycemia and finding new effective and affordable approaches to their treatment. Therefore, the aim of our work was to study the histological, ultramicroscopic, and histomorphometric features of reparative osteogenesis in rats with chronic hyperglycemia (CH), as well as to investigate the possibility of platelet-rich plasma (PRP) use in a fracture area in order to correct the negative effects of CH on reparative osteogenesis processes.研究对象和方法。该研究是在70只白色实验室大鼠进行，成熟雄性，将其分为以下几组：对照组，将动物用CH曝光，大鼠实验CH其用PRP施用到骨缺损的条件下创伤后胫骨缺陷，和动物的葡萄糖稳态和模拟CH确认的评估。使用Olympus BH-2显微镜（日本）进行光学显微镜检查。使用REM-102扫描型电子显微镜进行超微结构检查。采用SPSS-17软件进行统计分析。Results。两个星期后，并没有出现新的骨组织的动物形成与CH。只有在成骨修复的第30天的新形成的网织状骨组织为总面积再生的61.54％。这是小于基准值由22.89％（ )。关于成骨修复的第14天，一组动物用CH和PRP注射的在再生的区域由结缔组织的由68.94％（高于动物用CH少4.94％（ ))和编织骨组织由31.06％，（13.51％小于对照组（ ))。On the 30th day, the area of woven bone tissue in a regenerate of this group was less than that of the control group by 12.41% ( )。Conclusion。Thus, chronic hyperglycemia contributes to inflammation delay within the bone defect site, which makes the process of reparative osteogenesis more prolonged. The results of chronic hyperglycemia effect on bone regeneration are also impairment of osteogenic cell proliferation and shift of their differentiation towards the fibrocartilage regenerate formation. The PRP corrects the negative impact of chronic hyperglycemia on reparative osteogenesis, promoting more rapid inflammatory infiltrate removal from the bone defect site and osteogenic beam formation and remodeling of woven bone into lamellar membranous bone tissue.

1. Introduction

According to world statistics, musculoskeletal injuries rank second place among disability and mortality causes [1]。According to the forecast of Lopes et al., the annual fracture number in Europe will increase by 28% by 2025 (from 3.5 to 4.5 million cases) [2]。Today, according to the WHO, around 422 million people worldwide have diabetes mellitus (DM). In the next 25 years, experts predict the rise of DM incidence to 629 million, which is a major socioeconomic problem [3,4]。

In total, the number of musculoskeletal injuries in healthy persons’ clavicle fractures accounts for 17.5%; brachial bone, 8.2%; forearm bones, 60.8%; femur, 8.2%; and lower leg bones, 5.2% [5]。在糖尿病患者和糖耐量减低者，前臂骨折占21％;椎骨，18％;股骨，18％;并降低腿骨，19％[6]。Besides that, in DM patients, the proportion of delayed fragment consolidation, fissures, and false joints reaches from 8 to 32% compared to healthy individuals [7]。

近年来，自体血小板浓缩液已广泛应用于临床实践中的组织再生[8]。High efficiency of biological drugs based on platelet-rich plasma (PRP) in sports injury treatment and during operations on joints and bones in dentistry has been already proved [9,10]。然而，没有关于在从慢性高血糖症罹患或2型DM人的疗效和PRP使用可行性在长管状骨骨折的治疗不足的信息。

因此，在治疗糖尿病患者骨再生障碍，其患病率高，并发症数量，和困难的不尽人意的后果需要进一步研究和在慢性高血糖，并寻找新的有效和负担得起的方法来他们的治疗修复骨形成机制更深入的了解。

The aim of this work was to study the histological, ultramicroscopic, and histomorphometric features of reparative osteogenesis in rats with chronic hyperglycemia, as well as to investigate the possibility of PRP use in a fracture area to correct the negative effects of chronic hyperglycemia on reparative osteogenesis processes.

2。材料和方法

70 white laboratory male rats (age—7-9 months) were used for experimental study. All animals were divided into the following groups: group I—control (animals with traumatic injury of the tibia) (20 rats); group II—animals with experimental CH and traumatic injury of the tibia (20 rats); group III—rats with experimental CH and traumatic injury of the tibia, which received PRP into the area of tibia fracture (20 rats); and group IV—animals for glucose homeostasis evaluation and confirmation of modeled CH (10 rats).

所有的动物检查它们的运动活动及外壳的条件。然后，大鼠进行为期两周的隔离。实验动物根据一般伦理的实验动物的原则（基辅，2001年）是在状态，赫尔辛基（2000），和欧洲公约宣言用于试验和其他科学目的（斯特拉斯堡，1985年）的保护脊椎动物。伦理和道德研究的过程中不受侵犯。大鼠在恒定温度（24-25℃），湿度下的动物饲养室（ )%, and 12-hour dark-light cycle. Current cell cleaning was performed daily.

CH组动物II、III和IV是模型d as follows. For 2 weeks, rats have been consuming 10% aqueous fructose solution instead of drinking water. After that, intraperitoneal administration of streptozotocin (Sigma-Aldrich, USA) (40 mg/kg) and nicotinic acid (1 mg/kg) was performed once for each rat. Control group animals were administered single intraperitoneal injection of sterile citrate buffer. Following the streptozotocin administration, animals were kept under normal vivarium conditions on normal diet for 60 days.

On the 60th day after CH modeling, fasting blood glucose, insulin, glycosylated hemoglobin and C-peptide were determined in animals of group IV. Acquired data was used to confirm the CH presence.

Holey defect of both tibias was modeled in group I, II, and III rats for further investigation of reparative osteogenesis at micro- and ultrastructural levels. Surgery was performed in aseptic conditions under ketamine (8 mg/kg) and xylazine (3 mg/kg) anesthesia. 30 minutes before surgery, animals were intramuscularly administered a prophylactic dose of ampicillin (7.5 mg/kg). Preoperative preparation of the surgical field was performed by shaving the wool in the area of the anterior surface of the tibia and three times treating with 3% alcohol iodine solution.

Soft tissue sections of 0.8-1.5 cm long were made along the margo anterior line of the tibia. Using a portable dental drill (sterile boron (1.6 mm), low revolutions with cooling) formed port into the bone marrow in the middle third of the tibial diaphysis. Surgical wounds were closed with skin suture treated with 3% alcohol iodine solution.

In group I and II animals, the bone defect was left to heal under the blood clot. In group III rats, in order to correct possible negative CH influence on reparative osteogenesis, PRP (dose—0.5 ml) was introduced into the wound before suturing. For this, previously, 2 ml of blood from the lateral tail vein was collected into 4 ml vacuum containers containing 0.35 ml of 10% sodium citrate solution. The lost blood volume was immediately restored by sterile saline infusion. The selected blood was centrifuged for 20 min at a speed of 2,000 rpm. As a result, two blood component fractions were observed in the test tube: the lower dark red fraction (cellular components) and the upper straw yellow fraction (serum components). After that, the contents of the upper fraction and upper portion of the lower fraction were pipetted and transferred to another tube. The resulting material was centrifuged for 15 min at a speed of 2,000 rpm, which led to formation of two fractions: the lower, platelet-rich plasma and the upper, platelet-poor plasma. The contents of the lower fraction were transferred to a sterile tube, and the volume was adjusted to 1 ml with 10% calcium chloride solution [11]。The resulting solution was injected into the wounds of animals.

Animals were removed from the experiment by the overdose of thiopental anesthesia (4 mg/100 g body weight) on the 14th and 30th day after trauma (these stages of bone healing process correspond to cell proliferation and differentiation, bone formation, and its adaptive restructuring).

To study the microscopic structure, the prepared portions of the left tibia with a defect were fixed in 10% formalin solution. Then, demineralization was carried out in 5% aqueous Trilon B solution. Further samples were dehydrated in alcohols of increasing concentration and poured into paraffin. Then, using microtome MC-2, sections from obtained preparations were made (thickness of 4-6 μ米）。染色用苏木精 - 伊红进行。的Olympus BH-2显微镜（日本）用于光学显微镜。使用微电网，微波线路，和Digimizer计算软件（版本5.3.5）进行形态测定分析。测定了下列参数：炎症浸润面积（mm²), granulation tissue area (mm²), connective tissue area (mm²), bone tissue area (mm²), and cartilage area (mm²)。

For ultramicroscopic examination using scanning electron microscopy, the injured right tibia of rats was removed and fixed in 2.5% glutaraldehyde solution (in 0.2 M cacodylate buffer with at +4°C) and postfixed in 1% OsO4 solution (for 4 hours at +4°C). Dehydration was done using series of ethyl alcohol ascending concentrations. Before examination, the specimens were sputtered with gold in vacuum post “VUP-5.” After that, specimens were placed in a scanning electron microscope “REM 102” and photographed.

Statistical processing of all obtained numerical data was performed using SPSS (version 17.0, Chicago, IL, USA). Validation for normality of distribution was implemented using the Kolmogorov-Smirnov criterion. Data are presented as mean (M) and standard deviation (SD). The significance of differences between two groups was determined using Student’s criterion ( )。The difference was considered significant if the probability of chance ( )did not exceed 0.05 ( )。

3.结果

The blood metabolic parameters in groups I (control) and IV (CH) are presented in Table1。The fasting glucose ( )而糖化血红蛋白（ )were significantly higher in rats with CH compared to control animals. The insulin level was decreased in the CH group ( ),wherein the C-peptide amount was equal between two groups ( )。Thus, the obtained results confirm the presence of chronic hyperglycemia in experimental animals.

The histological and ultramicroscopic analysis of reparative osteogenesis in the control group on the 14th day revealed bone beams with clearly separated young lacunae and fibroreticular tissue with high cell density of fibroblastic and osteoblastic diferons. The electronic scans of the osteogenic beam surface revealed a large number of spherical and cubic osteoblasts with long and thin processes. In most trabeculae, osteoblasts were immobilized in their own matrix (Figure1(d))。

In animals with CH (group II), the defect area was filled with connective tissue. No complete wound cleaning from remnants of inflammatory infiltrate was observed. Local clusters of neutrophilic granulocytes, macrophages, lymphocytes, and adipocytes were localized in the central part of the defect (Figure1(b))。In addition, scattered groups of chondrogenic cells were sometimes found among cellular infiltrates and collagen fibers. Electron scans of bone regeneration samples revealed clusters of rounded cells with numerous thin processes. Single deformed erythrocytes and thin fibers have been detected in the intervals between cell conglomerates (Figure1（e）中)。

In group III, the ordering and transformation of connective tissue into osteoid beams were noted. Most intensively, this process took place near the maternal bone. The area of connective tissue with a large number of sinusoidal-type capillaries without signs of bone formation was observed in the defect center (Figure1(c))。所述再生表面的电子扫描显示与由无定形基质包围薄长过程成骨细胞（图图1（f）)。

Figure2shows the tissue-specific composition of regenerate structures of bone defects on the 14th day of experiment. The posttraumatic area in the control group consisted of connective tissue (55.44%) and woven bone tissue (44.56%), wherein residual signs of inflammation were retained in CH rats. Inflammatory cell infiltration occupied 8.37% of the entire osteoreparation zone and granulation tissue area, 9.99%. In contrast to control animals, the woven bone tissue formation on the 14th day in CH animals did not occur. Instead, cartilage islets were found (7.96% of the total regenerated area). The rest of the regenerated area was connective tissue (73.68%). In a group of animals, which received PRP injection, the defect site was filled with connective tissue (68.94%) and small bone trabeculae (31.06%). No residual signs of inflammation, granulation, and cartilage were detected.

在实验的第30天，对照组中的骨缺损几乎完全充满了新形成的骨组织。与此同时，新形成的编织骨组织重塑成层状膜状骨组织的过程已经开始。主动破骨细胞，骨梁周围观察。新的板层膜的骨组织包含与形成哈佛管完整骨单位。最密集，这些过程发生在靠近母体骨骼中。缺陷的中央区域仍然填充有编织骨组织（图图3（a）)。缺陷区域的电子扫描表明，其表面包覆有新骨形成，由许多经皮质血管开口和成骨细胞腔隙（图穿透3(d))。

In group II, different sizes of osteogenic beams were formed. Wide gaps were observed between the trabeculae. Their formation near maternal bone was more structured. There were areas with not fully formed and interconnected beams. In the central part of defect, they had uneven thickness and disorganized placement. In addition, there was local replacement of defect by cartilage in the environment of woven bone tissue (Figure3(b))。层状膜的形成bone tissue in this group did not occur. On electronic scans, the surface of the defect site had numerous intertrabecular gaps filled with cells and connective tissue. The fibrous structure of osteogenic beams was well visualized. Formation of complete periosteum of newly formed bone did not occur (Figure3(e))。

In group III animals, more than half of the tibial defect was filled with woven bone tissue. Initial foci of lamellar membranous bone formation with restored osteons and Haversian canals were observed only near maternal bone (Figure3(c))。扫描显微术揭示，缺陷中心区域的表面通过大量胶原链形成。浸渍在骨梁的骨样基质成骨细胞邻近母体骨骼进行观察。缺陷的中央区域中填充编织骨组织和结缔组织。关于骨膜，其对实验该组中的第30天形成才刚刚开始（图3(f))。

Tissue-specific composition of bone regenerate structures on the 30th day of experiment is shown on Figure4。In the control group, the defect area was filled with newly formed woven bone and lamellar membranous bone tissues (total area—84.44%). In rats with CH, the defect was filled with woven bone tissue (61.54%), connective tissue (28.22%), and cartilage tissue (10.24%). In group III, the formation of lamellar membranous bone had only just begun. The defect was mostly filled with woven bone (72.03%) and connective tissue (27.97%).

4。讨论

In recent years, approaches to treatment of musculoskeletal injuries have changed significantly. According to ideas of most current authors, the use of PRP is simple and affordable and is a minimally invasive way to obtain natural concentration of autologous mediators, such as insulin-like growth factor-1, basic fibroblast growth factor, platelet-derived growth factor, epidermal growth factor, vascular endothelial growth, and transforming growth factor beta, which play a major role in inflammatory response attenuation and necrotizing cell elimination and have the number of potential advantages over existing methods. The availability, simplicity, efficiency of the method, and the absence of allergic reactions open the prospects for its further study and wider use in clinical medicine [8–10,12,13]。

Research results report on high percentage (8 to 32%) of reparative osteogenesis disorders in type 2 DM patients and that hyperglycemia leads to decrease of proliferation and differentiation of cartilage and osteoblastic diferon cells involved in regeneration [14–17]。Our study revealed proliferation and differentiation disorders of osteoblastic diferon towards the formation of fibrocartilage regenerate in animals with CH.

Marin et al. and Hygum et al. showed that CH causes violation of coordinated action of signaling molecules and regulators of reparative osteogenesis, which leads to decreasing of osteoblast functioning, increasing of adipose tissue amount in regenerates, and significant inhibition of consolidation process [18,19]。我们osteoreparation阶段的分析显示，在消除炎症的动物与CH第一阶段的延迟。其结果是，在第14天，该组中包含再生脂肪细胞和淋巴细胞地方，白细胞浸润部分。

Insulin plays the important role in bone healing in DM patients through stimulation of bone matrix formation and increasing of collagen synthesis by osteoblasts. In vitro studies have identified decreasing of newly formed tissue ossification and impaired cartilage formation due to insulin deficiency. Researchers have found that collagen synthesis level in the fracture zone of DM rats decreased by 50-55%, which led to deterioration of mechanical properties of newly formed tissue [20]。In our study, we found impaired collagen structuring into osteoid beams and uneven formation and placement of cartilage in bone regenerates of animals with CH.

Dedukh和Sykal报道关于破骨细胞密度的不断增加，软骨区域的增加，骨，血管发生缺陷，和胶原蛋白和糖胺聚糖合成减值与DM的动物的骨再生区域软骨组织替代的病症[21]。Besides, the fibroreticular tissue area in bone regenerates of DM animals was significantly higher compared to that of control animals, which indicated the complication of reparative osteogenesis processes. Our morphometric analysis revealed that in animals with CH, the connective tissue area on the 14th day of reparative osteogenesis was higher by 18.24% ( )和12.66％，第30天（ ),相比于对照组。

相对于动物正常的血糖与CH动物有较长的修复骨。CH导致新血管生成损伤和炎症增强，其最终导致成骨细胞适当分布并抑制氧和营养物进入再生区的中断。此外，组织结构分解代谢紊乱和脂肪形成细胞的增殖在动物DM的再生骨组织中观察到。上述脂肪形成细胞增加脂肪组织中的断裂区域中的含量，这导致抑制骨片段整合[18,22]。我们的研究证实，成骨修复过程较长的动物CH。上osteoreparation处理的第30天，在动物用CH新形成的编织骨组织的面积为22.89％（ )less compared to control.

在III（动物用CH，其中注射PRP进入伤口）成骨上的第14天组，没有检测到炎症，造粒，和在形成的回收物软骨组织的残留迹象，不同于没有施用PRP动物。在第30天，编织骨组织在组III的大鼠的再生面积较大的相比，用CH动物，这些都没有施用的PRP。获得的数据证实，PRP处理降低受损组织肿胀，抑制急性炎症，并促进从改变相位到蓄热修复过程[快速移23]。

The abovementioned data also indicates antimicrobial activity of PRP, which is also shown in Bielecki et al. study [24]。Authors have analyzed the antibacterial effect of PRP in vitro. As a result, inhibition of Staphylococcus aureus and Escherichia coli growth and simultaneous induction of Pseudomonas aeruginosa growth were found. These data demonstrates different resistance of microorganisms to PRP. Authors believe that combination of inductive and antimicrobial PRP properties may improve the treatment outcomes of patients with infected fractures and false joints.

It should be said that there are few important limitations in our experiment, which did not allow us to evaluate the CH and PRP effect on bone regeneration more efficiently. Special staining methods with immunohistochemical reaction in order to recognize osteoblasts, endotheliocytes, and different types of leukocyte were not used. Thus, osteogenic cell amount, neoangiogenesis degree, and inflammation nature at the sites of bone regeneration were not accurately estimated. In addition, we have not applied PCR and immunohistochemistry methods for quantitative and qualitative assessment of molecular markers of bone regeneration (such as osteocalcin, bone sialoprotein, bone morphogenetic protein-2, and Runx2), which also did not allow us to describe the features of posttraumatic bone repair in different groups at a molecular level.

5. Conclusions

因此,慢性高血糖导致正无穷lammation delay within the bone defect site, which makes the process of reparative osteogenesis more prolonged. The results of chronic hyperglycemia effect on bone regeneration are also impairment of osteogenic cell proliferation and shift of their differentiation towards the fibrocartilage regenerate formation. The PRP corrects the negative impact of chronic hyperglycemia on reparative osteogenesis, promoting more rapid inflammatory infiltrate removal from bone defect site and osteogenic beam formation and remodeling of woven bone into lamellar membranous bone tissue.

Data Availability

用于支持研究结果的数据是可用的，请相应的作者。

利益冲突

All authors declare that there is no conflict of interests regarding the publication of this work.

Acknowledgments

这项研究是科学项目的一部分，“慢性高血糖状态下，下肢组织再生的分子遗传和形态特征”，由教育和科学部乌克兰（无。0117U003926）的支持。

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Copyright

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