Biomechanics of craniofacial sutures: orthopedic implications

Sutures are soft connective tissue articulations between craniofacial bones. Suture mechanics deals with patterns of mechanical stress experienced in sutures resulting from natural activities such as mastication and exogenous forces such as orthopedic loading. Patterns of sutural mechanical stress can be delineated readily as sutural strain using strain gages attached over the suture. In mastication, complex sutural strain patterns have been elucidated in a few species. Mechanical stresses are not transmitted in the skull as a continuing gradient, for different sutures are capable of redefining a propagating mechanical force as predominately tensile or compressive strain. Exogenous mechanical forces with engineering waveforms such as static and sine wave at different frequencies induce corresponding waveforms and rates of sutural strain, providing the basis for applying novel mechanical stimuli to engineer sutural growth. The available data on suture mechanics converge to a hypothetical theme that mechanical forces regulate sutural growth by inducing sutural mechanical strain. Various orthopedic therapies, including headgear, facemask, and functional appliances may induce sutural strain, leading to modification of otherwise natural suture growth.     

Indian hedgehog: a mechanotransduction mediator in condylar cartilage

Indian hedgehog (Ihh) is a critical mediator transducing mechanical signals to stimulate chondrocyte proliferation. To clarify the cellular signal transduction pathway that senses and converts mechanical signals into tissue growth in mandibular condyle, we evaluated Ihh expression and its relation to the kinetics of replicating mesenchymal cells in condylar cartilage during natural growth and mandibular advancement. Thirty-five-day-old Sprague-Dawley rats were fitted with functional appliances. Experimental animals with matched controls were doubly labeled with iododeoxyuridine and bromodeoxyuridine so that we could evaluate the cycles of the proliferative mesenchymal cells. Mandibular advancement triggered Ihh expression in condylar cartilage. A higher level of Ihh expression coincided with the increase of the replicating mesenchymal cells' population and the shortening of the turnover time. These findings suggested that Ihh acts as a mediator of mechanotransduction that converts mechanical signals resulting from anterior mandibular displacement to stimulate cellular proliferation in condylar cartilage.     

Cyclic mechanical strain regulates the PTHrP expression in cultured chondrocytes via activation of the Ca2+ channel

The association between mechanical stimulation and chondrocyte homeostasis has been reported. However, the participation of PTHrP (parathyroid-hormone-related protein) in the mechano-regulation of chondrocyte metabolism remains unclear. We determined whether mechanical stimulation of chondrocytes induces the expression of PTHrP and, further, whether the mechano-modulation of PTHrP is dependent on the maturational status of chondrocytes. Cyclic mechanical strain was applied to rat growth plate chondrocytes at the proliferating, matrix-forming, and hypertrophic stages at 30 cycles/min. Cyclic mechanical strain significantly increased PTHrP mRNA levels in chondrocytes at the proliferating and matrix-forming stages only. The induction of PTHrP was dependent on loading magnitude at the proliferating stage. Using specific ion channel blockers, we determined that mechano-induction of PTHrP was inhibited by nifedipine, a Ca2+ channel blocker. These results suggest that mechanical induction of PTHrP possibly provides the environment for greater chondrocyte replication and matrix formation that would subsequently affect cartilage formation.     

Mechanical strain delivers anti-apoptotic and proliferative signals to gingival fibroblasts

Physical forces play a critical role in the survival and proliferation of many cell types, including fibroblasts. Gingival fibroblasts are exposed to mechanical stress during mastication, orthodontic tooth movement, and wound healing following periodontal surgery. The aim of this study was to examine the effect of mechanical strain on human gingival fibroblasts (hGF). Cells were subjected to short-term (up to 60 min) and long-term (up to 48 hrs) 20% average elongation at 0.1 Hz. We monitored survival signaling by evaluating the phosphorylation status and localization of Forkhead box (FoxO) family members, which are mediators of apoptosis. We also examined strain-induced proliferation by measuring the level of proliferating cell nuclear antigen (PCNA). We observed that cyclic strain caused the phosphorylation and retention in the cytoplasm of FoxO family members. Moreover, mechanical strain resulted in increased ERK kinase phosphorylation and PCNA expression. In conclusion, cyclic strain delivers anti-apoptotic and proliferative stimuli to hGF.     

永生化髁突软骨细胞的血清依赖性生长实验

目的 研究永生化髁突软骨细胞（Immortalizedmandibularcondylarchondrocyte,IMCC）的血清依赖性生长特征。方法 将IMCC和原代培养的躲突软骨细胞（Mandibularcondylarchondrocyte,MCC）接种于96孔板,用MTT法比较两种细胞在无血清培养基,含5％、10％和20％血清培养基中的生长情况。结果 IMCC和MCC均不能在无血清培养基中生长,但可在含5％血清的培养基中生长,其增殖速度明显低于在10％和20％两种血清浓度中细胞的生长速度,而10％和20％两种血清浓度中细胞的生长速度则较接近。IMCC的生长速度快于MCC。结论 IMCC保留了MCC的血清依赖性生长特性,其增殖速度快于MCC,不属于恶性转化细胞。     

Laboratory stresses and tractional forces on the TMJ disc surface

The etiology of degenerative disease of the TMJ may involve fatigue produced by surface tractional forces and compressive stresses. This study tested the time-dependent effects of compressive loading and stress-field translation on TMJ disc-surface tractional forces and stresses. In laboratory experiments with 50 porcine discs, an acrylic indenter imposed 10 N static loads for 10 and 60 sec, followed by translation of the loaded indenter along the mediolateral axis of the disc. Maximum tractional forces were found to occur following 60 sec of static loading (p < 0.001), and increased with translation velocity (R(2) = 0.73); whereas maximum compressive stresses occurred after 10 sec of static loading (p < 0.001). Overall, the results were consistent with current mechanical theories of the time-dependent effects of compressive loading of cartilage.     

Design and performance of a bioreactor system for mechanically promoted three-dimensional tissue engineering

There is currently considerable interest in increasing the response of mesenchymal cells to physical forces, and numerous loading devices have been used to increase the formation of skeletal tissue in vivo and in vitro. We have developed a bioreactor system to apply cyclic strains on three-dimensional specimens over a range of 0-20,000 mustrain. The piezoelectric-driven mechanism allows the precise adjustment and control over load-related deformations of tissue, as shown by finite-element calculations of deformation of a collagen gel under load. We present the design of the bioreactor and its performance in specimens of tissue containing activated osteoblasts and chondrocytes. Biaxial tissue straining at 2,000 mustrain led to a substantial increase in the number of both types of cell compared with unstimulated controls. The synthesis of cell-specific extracellular matrix proteins increased when physiological loads (2,000 mustrain) were applied in the bioreactor, whereas higher deformations (20,000 mustrain) resulted in a reduction in proliferation and differentiation of cells. The mechanisms whereby mechanical stimulation leads to a defined cell reaction are not known, but the application of physiological micromovements in extracorporeal tissue chambers is a promising approach to the formation of hard tissue.     

Abfractions: A New Classification of Hard Tissue Lesions of Teeth

Due to the stresses resulting from biomechanical loading forces exerted on the teeth (static, as in swallowing and clenching or cyclic, as in chewing), both enamel and dentin can chip or break away. This loss of tooth substance, which shall be termed Abfraction , is dependent on the magnitude, duration, direction, frequency, and location of the forces. These abfractive lesions are caused by flexure and ultimate material fatigue of susceptible teeth at locations away from the point of loading. Clinical observation of a variety of enamel and dentin lesions due to the shapes, sizes, loci, and frequency warrants a new and distinct classification.     

Abfractions: a new classification of hard tissue lesions of teeth

Due to the stresses resulting from biomechanical loading forces exerted on the teeth (static, as in swallowing and clenching or cyclic, as in chewing), both enamel and dentin can chip or break away. This loss of tooth substance, which shall be termed Abfraction, is dependent on the magnitude, duration, direction, frequency, and location of the forces. These abfractive lesions are caused by flexure and ultimate material fatigue of susceptible teeth at locations away from the point of loading. Clinical observation of a variety of enamel and dentin lesions due to the shapes, sizes, loci, and frequency warrants a new and distinct classification.     

The PI3K/Akt signalling pathway may play an internal role related to abnormal condylar growth: a preliminary study

Developmental deformity of the mandible is one of the most common craniofacial malformations and is closely related to abnormal condylar growth. In this study, the role of PI3K/Akt signalling in the regulation of chondrocyte proliferation and hypertrophic differentiation in the condylar cartilage was studied. Immunohistochemical staining was used to investigate the expression of PI3K and p-Akt in the rat condyle cartilage. Rat condylar chondrocytes were cultured for the investigation of chondrocyte proliferation and hypertrophic differentiation when PI3K/Akt was inhibited. In addition, organ culture of the rat mandibular condyle was performed to evaluate the condyle cartilage growth while PI3K/Akt was inhibited. PI3K-positive cells and p-Akt-positive cells showing cytoplasmic staining were found to be present in the condylar cartilage. Reduced cell proliferation was observed in the culture of rat condylar chondrocytes when PI3K/Akt was inhibited; however, the hypertrophic differentiation level was increased. The proliferative zone thickness of condylar cartilage in the experimental group was less than that in the control group (P = 0.00185), but the hypertrophic zone was greater than that in the control group (P = 0.01048). PI3K/Akt signalling exerts opposite influences on chondrocyte proliferation and hypertrophic differentiation of the condylar cartilage, and these data suggest that PI3K/Akt is a potential intracellular regulation signal pathway in condylar cartilage development.     

静张应力对大鼠髁突软骨细胞增殖效应调节研究

目的　探讨静张应力环境对髁突软骨细胞增殖效应的影响。方法　将第4代大鼠下颌髁突软骨细胞在细胞膜式张应力施加装置上培养,检测不同血清浓度及持续不同的静张应力(5 kPa、10 kPa)在0~12 h内对髁突软骨细胞增殖活性的影响。结果　低血清培养基中,随培养时间的延长,硅胶膜上髁突软骨细胞的增殖活性受到抑制;短期(2h)的静张应力(5 kPa、10 kPa)对髁突软骨细胞细胞周期的调控影响不大;0~10 h内5 kPa静张应力组软骨细胞增殖指数随着张应力作用时间的延长不断上升,增殖指数峰值位于10 h处;0~8 h内10 kPa静张应力组软骨细胞增殖指数随着张应力作用时间的延长不断上升,增殖指数峰值位于8 h处;5 kPa静张应力组较10 kPa静张应力组对髁突软骨细胞具有更大的促增殖作用。结论　静张应力可以调节髁突软骨细胞的增殖活性。     

软骨细胞增殖与凋亡在人重组白细胞介素-1β介导的颞下颌关节炎症损伤中的意义

目的:探讨细胞增殖与凋亡在人重组白细胞介素-1B介导的颞颌关节炎症损伤中的作用。方法:选用SD大鼠颞下颌关节腔内反复多次注射rhIL-1B,造成炎症损伤,利用免疫组织化学染色及原位末端标记法,观察炎症损伤过程中髁突软骨细胞增殖及凋亡的变化。结果:注射后1~30 d,实验侧髁突软骨增殖层PCNA阳性指数均较对照侧低,以1~7 d最明显;实验侧髁突软骨细胞凋亡数较对照侧多,主要分布于软骨增殖层及前肥厚层中,以1~7 d 最明显,15 d后凋亡细胞数明显减少,仅见个别凋亡细胞散在分布于软骨浅层。结论:rhIL-1B关节内注射不仅使髁突软骨细胞增殖受抑制,而且细胞凋亡也可能是rhIL-1B致关节损伤的途径之一,细胞增殖与凋亡协同参与了颞下颌关节炎症损伤的病理过程。     

Force-induced rapid changes in cell fate at midpalatal suture cartilage of growing rats.

The application of expansional force induces replacement of the cartilaginous tissue with bone at the midpalatal suture of growing rats. We examined the early cellular events evoked by force by analyzing the expression of proliferating cell nuclear antigen (PCNA), an operational marker of cell proliferation, and of several bone matrix proteins. A rectangular orthodontic appliance was set between the right and left upper molars of four-week-old rats, with 50 g of initial expansional force. Two days after application of the force, the pre-existing cartilage was separated laterally. Mesenchymal cells with stretched shapes were arranged parallel to the expansional force and filled the center of the suture. Only a few of these stretched cells exhibited nuclear accumulation of PCNA. In contrast, many polygonal mesenchymal cells distributed along the inner lateral side of the cartilaginous tissue exhibited strong immunoreactivity for PCNA. Localization of alkaline phosphatase activity overlapped into this proliferating cell zone. Nascent extracellular matrix under the proliferating cells was positive for osteocalcin, indicating commencement of active bone formation. These findings indicated that, among mesenchymal cells subjected to expansional forces, only cells located on the inner side of the cartilaginous tissue proliferate and differentiate into osteoblasts. In agreement with rapid bone growth progression, apoptosis was also observed in the zone of proliferating cells, as measured by TdT-mediated dUTP-biotin nick end labeling (TUNEL) assays.     

骨保护素对去卵巢小鼠髁状突骨和软骨的影响

目的观察外源性骨保护素(osteoprotegerin,OPG)对去卵巢小鼠髁状突骨及软骨的影响。方法4月龄雌性未孕小鼠30只,其中20只行双侧卵巢摘除术,10只行假手术(A组)。4周后将双侧卵巢摘除20只随机分为B、C两组各10只,B组予安慰剂,C组皮下注射OPG 10 mg/kg,每周2次。治疗2个月后,测定各组动物髁状突的骨小梁面积、数量,软骨层的厚度及软骨层内部各层细胞的数量等。结果B组髁状突的骨小梁面积、数量较A组明显降低。C组髁状突的骨小梁面积、数量较A、B组明显增加,软骨层厚度增加、软骨层内部肥大细胞层较A、B组明显增厚。结论小鼠去卵巢后可成功建立髁状突骨质疏松模型;OPG对去卵巢小鼠髁状突有抑制骨吸收作用,并可能促进软骨细胞增殖,促使软骨细胞向成骨过渡。     

不同加载频率拉应力对髁突软骨细胞增殖分化的影响

目的:探讨不同加载频率的拉应力对髁突软骨细胞增殖、分化的影响。方法:体外分离2周龄新西兰兔髁突软骨细胞,对细胞施加2 h/d,为期3 d的不同频率(0 Hz、0.1 Hz、0.5 Hz、1 Hz)的拉应力。采用CCK8检测细胞增殖水平,实时荧光定量RT-PCR检测Sox9、Runx2、VEGF的表达。结果:细胞增殖水平在静止性(0 Hz)组显著高于周期性加力(0.1~1 Hz)组和对照组;Sox9、Runx2、VEGF表达在0.5 Hz组和1 Hz组显著高于静止性(0 Hz)组和对照组。结论:不同加载频率拉应力对髁突软骨细胞增殖分化有差异性影响。     

Growth and development: hereditary and mechanical modulations

Growth and development is the net result of environmental modulation of genetic inheritance. Mesenchymal cells differentiate into chondrogenic, osteogenic, and fibrogenic cells: the first 2 are chiefly responsible for endochondral ossification, and the last 2 for sutural growth. Cells are influenced by genes and environmental cues to migrate, proliferate, differentiate, and synthesize extracellular matrix in specific directions and magnitudes, ultimately resulting in macroscopic shapes such as the nose and the chin. Mechanical forces, the most studied environmental cues, readily modulate bone and cartilage growth. Recent experimental evidence demonstrates that cyclic forces evoke greater anabolic responses of not only craniofacial sutures, but also cranial base cartilage. Mechanical forces are transmitted as tissue-borne and cell-borne mechanical strain that in turn regulates gene expression, cell proliferation, differentiation, maturation, and matrix synthesis, the totality of which is growth and development. Thus, hereditary and mechanical modulations of growth and development share a common pathway via genes. Combined approaches using genetics, bioengineering, and quantitative biology are expected to bring new insight into growth and development, and might lead to innovative therapies for craniofacial skeletal dysplasia including malocclusion, dentofacial deformities, and craniofacial anomalies such as cleft palate and craniosynostosis, as well as disorders associated with the temporomandibular joint.     

静张应力与TGF-β_1对大鼠髁突软骨细胞增殖效应调节初步研究

目的　探讨转化生长因子-β1(TGF-β1)在静张应力环境下对髁突软骨细胞增殖效应的影响。方法　(1) 将传代培养至第4代大鼠下颌髁突软骨细胞在12孔培养板上培养,采用流式细胞仪测定加入不同浓度TGF-β1(0·1、1 、10 ng/ml) 0、6、12、18、24 h后,对髁突软骨细胞增殖活性(PI值)的影响。(2)将第4代大鼠下颌髁突软骨细胞在细胞膜式张应力施加装置上培养,检测不同浓度TGF-β1及5 kPa静张应力联合应用0、6、12 h后,对髁突软骨细胞增殖活性 (PI值)的影响。结果　不同浓度TGF-β1(0·1、1、10 ng/ml)在模拟生理环境下以剂量依赖型的方式整体促进了髁突软骨细胞增殖效应,对髁突软骨细胞的增殖活性调节作用主要在12~18 h段,增殖峰值在18 h左右;在5 kPa静张应力联合TGF-β1刺激下培养较TGF-β1的单独效应具有更强的促髁突软骨细胞增殖作用。结论　静张应力联合TGF-β1可以调节髁突软骨细胞的增殖活性。     

Engine-driven preparation of curved root canals: measuring cyclic fatigue and other physical parameters

An increasing number of engine-driven rotary systems are marketed to shape root canals. Although these systems may improve the quality of canal preparations, the risk for instrument fracture is also increased. Unfortunately, the stresses generated in rotary instruments when shaping curved root canals have not been adequately studied. Consequently, the aim of an ongoing project was to develop a measurement platform that could more accurately detail physical parameters generated in a simulated clinical situation. Such a platform was constructed by fitting a torque-measuring device between the rotating endodontic instrument and the motor driving it. Apically directed force and instrument insertion depth were also recorded. Additional devices were constructed to assess cyclic fatigue and static fracture loads. The current pilot study evaluated GT rotary instruments during the shaping of curved canals in plastic blocks as well as "ISO 3630-1 torque to fracture" and number of rotations required for fatigue fracture. Results indicated that torques in excess of 40 Nmm were generated by rotary GT-Files, a significantly higher figure than static fracture loads (less than 13 Nmm for the size 20. 12 GT-File). Furthermore, the number of rotations needed to shape simulated canals with a 5 mm radius of curvature in plastic blocks was 10 times lower than the number of rotations needed to fracture instruments in a "cyclic fatigue test". Apical forces were always greater than 1 N, and in some specimens, scores of 8 N or more were recorded. Further studies are required using extracted natural teeth, with their wide anatomical variation, in order to reduce the incidence of fracture of rotary instruments. In this way, the clinical potential of engine-driven rotary instruments to safely prepare curved canals can be fully appreciated.     

Role of epidermal growth factor and its receptor in mechanical stress-induced differentiation of human periodontal ligament cells in vitro

The periodontal ligament (PDL) contains precursor cells for osteoblasts and cementoblasts. It has been shown that epidermal growth factor (EGF) inhibits dexamethasone-induced differentiation and up-regulates EGF-receptor (EGF-R) expression, whereas EGF-R is down-regulated in the course of differentiation. Thus it was suggested that EGF and its receptors act as a negative regulator of osteoblastic differentiation in PDL cells. In order to investigate further this hypothesis, human PDL cells were now used to elucidate the role of EGF and EGF-R in their proliferation and differentiation under mechanical stress-loaded conditions in vitro, as the PDL regularly receives mechanical stress from occlusal forces. As a model of mechanical stress, a cyclic stretch of 9 or 18% elongation was applied to the cells with a Flexercell cell-strain unit system. Alkaline phosphatase activity and osteocalcin mRNA expression were significantly induced by loading cyclic stretch for more than 4 days, whereas stretch slightly inhibited cell proliferation. Visualization of the actin stress fibres of the cells by rhodamine phalloidin revealed that approx. 10% of the total number of cells had become aligned perpendicularly to the direction of the stretch. The effects of stretch on alkaline phosphatase activity and cell proliferation were totally abolished by the presence of 10 ng/ml EGF. Western blotting of EGF-R protein demonstrated that stretch-induced differentiation accompanied the decreased expression of EGF-R protein in the cells. However, the amount of tyrosine-phosphorylated EGF-R upon EGF stimulation was restored to the control level in stretched cells. These results suggest that the EGF/EGF-R system acts as a negative regulator of differentiation of PDL cells regardless of the type of differentiation stimuli. Also, interaction between mechanical stress and the EGF/EGF-R system may participate in the osteoblastic differentiation of PDL cells and thereby regulate the source of cementoblasts and osteoblasts.