Platelet-rich plasma induces annulus fibrosus cell proliferation and matrix production

Purpose

Platelet-rich plasma (PRP) contains growth factors and creates a 3D structure upon clotting; PRP or platelet lysate (PL) might be considered for annulus fibrosus (AF) repair.

Methods

Bovine AF cells were cultured with 25 % PRP, 50 % PRP, 25 % PL, 50 % PL, or 10 % FBS. After 2 and 4 days, DNA, glycosaminoglycan (GAG), and mRNA levels were analyzed. Histology was performed after injection of PRP into an AF defect in a whole disc ex vivo.

Results

By day 4, significant increases in DNA content were observed in all treatment groups. All groups also showed elevated GAG synthesis, with highest amounts at 50 % PL. Collagen I and II expression was similar between groups; aggrecan, decorin, and versican expression was highest at 25 % PL. Injection of PRP into the AF defect resulted in an increased matrix synthesis.

Conclusions

Platelet-rich preparations increased the matrix production and cell number and may therefore be considered to promote AF repair.     

单层组织工程纤维环取向纳米纤维支架的构建和初步验证

目的 使用静电纺丝技术构建取向纳米纤维支架,观测其结构,测试其力学特点,观察支架与细胞之间的相互作用,探讨其作为纤维环组织工程支架的可能性.方法 使用高速滚轴作为收集装置制备聚左旋乳酸聚己内酯[poly（lactic acid-co-caprolactone,P（LIA-CL）]取向纳米纤维支架.以P（LLA-CL）无规纳米纤维支架作为对照,观察取向纳米纤维支架的纤维直径、角度分布、孔径等表观指标;测试其拉伸力学行为;体外接种骨髓间充质干细胞观察其体外增殖和生长形态情况.结果 P（LLA-CL）取向纳米纤维支架纤维取向性良好,平行于纤维方向上的拉伸力学性能得到明显增强.接种细胞后,支架可促进细胞增殖,并诱导细胞沿着纤维方向拉伸并定向排列.结论 取向纳米纤维支架的力学强度在取向方向上得到了明显增强,具有和单层纤维环相似的各向异性力学特点,并可以诱导细胞沿纤维方向定向排列,细胞分布与纤维环细胞排列方式相似,可用于纤维环组织工程研究.     

Theoretical model and experimental results for the nonlinear elastic behavior of human annulus fibrosus.

The physical properties of the annulus fibrosus are critical to the intervertebral disc's biomechanical function; alterations with degeneration and aging can contribute directly to joint dysfunction and pain. A constitutive model that links the mechanical structure of the annulus to its material properties is important for many bioengineering purposes. To this end, we developed a strain energy function with separate terms to represent the matrix, the fibers, and the interactions between the constituents. Additionally, we measured the tensile and compressive stress-strain response of the annulus in the circumferential direction. We simultaneously applied the strain energy function to these new data and to data from a wide range of experimental protocols reported in the literature. By choosing experimental protocols that use an unloaded reference configuration, we developed a comprehensive formulation for the multiaxial annular elastic behavior. As a partial validation, this formulation predicted experimental results that were not included in model parameter specification. We anticipate that this constitutive formulation will be useful for computational simulations of the disc's biomechanical response and for elucidating structure-function relationships of the annulus fibrosus.     

Modulation of annulus fibrosus cell alignment and function on oriented nanofibrous polyurethane scaffolds under tension

Background contextAnnulus fibrosus (AF), a component of the intervertebral disc (IVD), is always under tension in vivo, a condition that must be taken into consideration when tissue engineering an IVD. Loss of the tensile forces has been implicated in the pathogenesis of disc degeneration characterized by mechanical and structural breakdown of the AF.PurposeIn this study, we hypothesize that tensile forces modulate cellular and molecular behavior of AF cells grown on nanofibrous scaffolds in vitro.Study design/settingBovine AF cells were seeded onto strained electrospun-aligned nanofibrous polycarbonate urethane (PU) scaffolds. Tension was either maintained throughout the culture duration (monotonic) or removed after 24 hours (relaxed).MethodsThe effect of tension on AF cells cultured on PU scaffolds was evaluated over 7 days by scanning electron microscopy, biochemical assays, immunofluorescence microscopy, and quantitative polymerase chain reaction.ResultsCells grown on the relaxed scaffold were significantly more proliferative, synthesized more collagen and had increased collagen type I and TGFβ-1 gene expression; however these cells were not as aligned as were the cells and matrix on monotonic strained scaffolds. The alignment of AF cells grown on monotonic scaffolds correlated with significantly greater scaffold elastic modulus on day 7. Additionally, the cellular response to the change in strain was delayed by 3 to 5 days after tension release, which correlated with the time at which changes in scaffold length were detected.ConclusionsThis study demonstrated that AF cells respond at the molecular and cellular level to the changes in matrix/scaffold tension. This suggests that it may be necessary to determine the optimal elastic modulus and applied tensile forces to tissue engineer an AF that mimics the native tissue. Furthermore, this study provides insight into how changes in tensile forces may lead to changes in the AF cell function.     

Enhanced prolylhydroxylase activity in the posterior annulus fibrosus of canine intervertebral discs following long-term running exercise

Summary The effect of long-term exercise on the intervertebral disc collagen concentration (hydroxyproline), collagen-synthesizing enzymes (prolyl-4-hydroxylase, PH, and galactosyl-hydroxylysyl glucosyltransferase, GGT) and hydroxypyridinium crosslinks was studied in ten female beagle dogs. The dogs were run on a treadmill for 1 year starting at the age of 15 weeks. The daily running distance was gradually increased to 40 km, which distance the dogs ran for the final 15 weeks. Ten untrained dogs from the same breeding colony served as controls. The nucleus pulposus and anterior and posterior halves of the annulus fibrosus of C2–3, T10–12, L4–5 disc segments were analysed. Crosslinks were measured from the anterior annulus fibrosus of the T10–11 disc. Hydroxyproline and hydroxypyridinium concentrations remained similar in both groups. PH and GGT were significantly elevated by running in the posterior annulus fibrosus of the thoracic and lumbar discs and in the lumbar nucleus pulposus. In the thoracic nucleus pulposus GGT was reduced significantly. The results suggest activated collagen metabolism in the posterior annulus fibrosus of the thoracic and lumbar discs as a result of locally increased strains on the spine.The research was carried out at the Department of Anatomy, University of Kuopio     

腰椎椎间盘后方高信号区的临床意义

 目的 通过 CT、MRI及组织学分析,探讨腰椎间盘后方高信号区（high-intensity zone,HIZ）的病理学性质。 方法 选取下腰痛患者中其 MRI T2 加权像存在局限性 HIZ患者 41例,男 23例,女 18例;年龄 33~50岁,平均年龄 38岁。按照 MRI T1 加权像 HIZ的特点分为两组,T1 加权非高信号组（29例）即 T2 加权像呈高信号,T1 加权像呈低或等信号;T1 加权高信号组（12例）即 T1、T2 加权像均呈高信号。所有病例均同时进行 X线和 CT检查,T1 加权非高信号组中 26例进行了椎间盘造影以及疼痛激发试验。T1 加权非高信号组 15例、T1 加权高信号组 7例接受了手术治疗,手术方式分别为经椎间孔椎体间融合术或后路椎体间减压融合术,并对术中切取的 HIZ区域标本进行 HE染色和（或）免疫组织化学染色进行观察。 结果 T1 加权非高信号组患者 X线、CT及 MR检查均显示椎间盘退变,26例进行椎间盘造影以及疼痛激发试验者中 21例造影阳性及疼痛复制,并显示纤维环破裂,其中 15例阳性者接受手术治疗;5例阴性。术中切取的 HIZ区域标本 HE染色显示为纤维环破裂伴随肉芽组织长入,CD34染色可见 HIZ区域内新生毛细血管,CD68见 HIZ区域内多量染色阳性的巨噬细胞,而 HIZ外区域表达较弱。而 T1 加权高信号组患者椎间盘在 CT上显示为后方纤维环钙化或骨化,HE染色可见明显的钙化灶和钙化的潮汐线形成。结论 腰椎间盘后方 HIZ,在 T2 加权像的局限性高信号、T1 加权像呈局限性较低或等信号意味着通常所指的纤维环破裂伴随肉芽组织长入;而 T2、T1 加权像均呈高信号可能是钙化或骨化灶,因此,传统 HIZ 的概念可能需要进行一定的修正,结合 CT 检查可能更有意义。     

椎间盘灌注培养条件下压应力频率对纤维环细胞基质合成的影响

目的:观察压应力频率对椎间盘纤维环(AF)细胞基质合成的影响。方法:获取4~6月龄巴马香猪胸腰段椎间盘72个后,随机分为无应力对照组和应力组(分别为0.1Hz组、0.5Hz组、1Hz组、3Hz组、5Hz组),每组12个样本,将各组椎间盘置于生物反应器中进行灌注培养。无应力对照组不施加应力刺激,各应力组每天施加对应频率的应力刺激2h。灌注培养5d后分离全层纤维环组织行HE染色、阿利新蓝染色、胞外基质基因Aggrecan、CollagenⅠ、CollagenⅡ定量PCR、糖胺多糖(GAG)含量和羟脯氨酸(HYP)含量的检测。结果:培养5d后,各组纤维环组织仍保持天然的层状叠加结构。与对照组比较时,除5Hz组外其他应力组(0.1Hz、0.5Hz、1Hz、3Hz)纤维环组织阿利新蓝染色强度增强、各胞外基质基因的表达得到维持(0.1Hz)或上调(0.5Hz、1Hz、3Hz)、GAG和HYP的含量也维持在同等水平(0.1Hz)或显著升高(0.5Hz、1Hz、3Hz)。然而各应力组组间比较时,5Hz组纤维环组织阿利新蓝染色强度、胞外基质基因的表达水平及GAG和HYP的含量比其他应力组(0.1Hz、0.5Hz、1Hz、3Hz)都下调(P0.05)。结论:椎间盘体外灌注培养条件下,压应力刺激频率影响纤维环细胞的基质合成,低频率(0.1~3Hz)的应力刺激增加基质合成,高频率(5Hz)的应力刺激减少基质合成。     

The internal mechanical properties of cervical intervertebral discs as revealed by stress profilometry

Extensive anatomical differences suggest that cervical and lumbar discs may have functional differences also. We investigated human cervical discs using “stress profilometry”. Forty-six cadaveric cervical motion segments aged 48-90 years were subjected to a compressive load of 200 N for 20 s, while compressive ‘stress’ was recorded along the posterior-anterior midline of the disc using a pressure transducer, side-mounted in a 0.9 mm diameter needle. Stress profiles were repeated with the transducer orientated horizontally and vertically, and with the specimen in neutral, flexed and extended postures. Profiles were repeated again following creep loading (150 N, 2 h) which simulated diurnal water loss in vivo. Stress profiles were reproducible, and measured “stress” at each location was proportional to applied load. Stress profiles usually showed a hydrostatic nucleus with regions of higher compressive stress concentrated anteriorly in flexion, and posteriorly in extension. Stress concentrations increased in degenerated discs and following creep. Some features were unique to cervical discs: many showed a stress gradient across their central regions, even though vertical and horizontal stresses were equal to each other, and stress concentrations in the posterior annulus were generally small. Central regions of many cervical discs show the characteristics of a “tethered fluid” which can equalise stress over small distances, but not large. This may be attributable to their fibrous texture. The small radial diameter of the cervical posterior annulus may facilitate buckling and thereby prevent it from sustaining high compressive stresses.     

经Wiltse入路Quadrant通道下椎间盘切除术并纤维环修复术治疗单节段腰椎椎间盘突出症

目的 探讨经Wiltse入路Quadrant通道下椎间盘切除术并纤维环修复术治疗单节段腰椎椎间盘突出症(LDH)的临床疗效.方法 2016年1月—2018年9月收治单节段LDH患者82例,其中采用经Wiltse入路Quadrant通道下椎间盘切除术治疗47例(对照组),采用经Wiltse入路Quadrant通道下椎间盘...     

氧浓度及Notch通路对大鼠纤维环细胞增殖和细胞周期的影响

目的检测不同氧浓度条件下椎间盘纤维环细胞Notch信号通路相关分子的表达变化,明确参与调节纤维环细胞增殖的相关靶基因。方法体外分离、培养大鼠纤维环细胞,运用CCK-8细胞活力检测试剂盒、流式细胞仪检测不同氧浓度培养条件下Notch信号通路抑制剂L685458(2μmol/L、4μmol/L、8μmol/L)对纤维环细胞增殖及细胞周期的影响;荧光定量RT-PCR检测不同氧浓度培养条件下纤维环细胞Notch信号表达水平变化。结果 CCK-8结果显示与常氧条件相比,低氧培养纤维环细胞时所测吸光度值明显升高;无论常氧或低氧条件,加入Notch抑制剂干预后所测吸光度值明显降低;流式细胞仪结果显示与常氧条件相比,低氧培养纤维环细胞处于S/G2期细胞所占百分率明显升高,加入Notch抑制剂干预后处于S/G2期细胞所占百分率明显降低。低氧干预8~24 h后,Notch3、Notch4、Delta-like1、Delta-like3、Hes1、Hes5、Hes7 mRNA都有不同水平上调,Hey2 mRNA表达水平下调。结论低氧可以通过上调Notch信号通路的表达水平促进纤维环细胞增殖,Notch信号可能作为一个研究靶点用于延缓椎间盘退变的过程。     

腰椎间盘前缘与后缘高信号区的临床对照研究

目的 通过与腰椎间盘后缘高信号区(HIZ)比较,探讨前缘HIZ的发生规律、分布及临床意义.方法 根据HIZ的诊断及定位,将610例有完整临床资料的腰椎MRI,依据有无HE及其发生部位分为对照组(无HIZ),前缘组,后缘组和前后缘组.统计总样本中HIZ发生率,对比分析各组性别比例、年龄、体重、腰痛发生率及HIZ分布规律.结果 610例患者中,对照组315例(51.6%),前缘组95例(15.6%,119个椎间盘),后缘组159例(26.1%,189个椎间盘),前后缘组41例(6.7%,96个椎间盘).前、后缘组发生率间存在显著的统计学差异(P<0.01).各组的性别构成和体重载明显差异(P>0.05),年龄间存在显著的统计学差异(P<0.01,对照组<后缘组<前后缘组<前缘组).前缘组多见于L1,2～L4,5,后缘多位于L3,4～L5S1对照组、前缘组、后缘组和前后缘组腰痛发生率分别为40.0%,52.6%,55.4%和65.8%,对照组明显低于其他三组(P<0.05),后三组间差异无统计学意义(P>0.05).结论 与纤维环后缘HIZ相比,前缘HIZ发生率较低,患者年龄较大,发生节段较高,与腰痛症状亦明显相关.     

Cyclic stretch-induced apoptosis in rat annulus fibrosus cells is mediated in part by endoplasmic reticulum stress through nitric oxide production

Various mechanical stresses in vivo induce disc cell apoptosis and intervertebral disc (IVD) degeneration, but the underlying molecular mechanism is not fully known. The aim of this study was to investigate the role of endoplasmic reticulum stress in cyclic stretch-induced apoptosis of rat annulus fibrosus (AF) cells. Flexercell Tension Plus system was used to apply cyclic stretch to rat annulus fibrosus cells at a frequency of 0.5 Hz with 20% elongation for 12, 24, 36, or 48 h. Apoptosis was detected by flow cytometry, and nuclei morphologic changes were visualized by Hoechst 33258 staining and caspase-8, 9 activity assays. The expression of the markers of endoplasmic reticulum stress including CHOP, GRP78, and caspase-12 were determined by RT-PCR and Western blot. Mitochondrial membrane potential change was observed by JC-1 staining in situ. In addition, the levels of the nitric oxide (NO) were determined with the Griess reaction and fluorescence staining. The results indicated that cyclic stretch at a frequency of 0.5 Hz with 20% elongation-induced apoptosis in rat AF cells. Prolonged exposure of the unphysiologically cyclic stretch to AF cells caused NO overproduction, up-regulation of endoplasmic reticulum stress markers including CHOP, GRP78, and caspase-12, depolarization of mitochondria and activation of caspase-9. However, cyclic stretch at this level had no effect on caspase-8 activity. In addition, specific inhibitor of caspase-12 (Z-ATAD-FMK) and caspase-9 (Z-LEHD-FMK) partly suppressed cyclic stretch-induced AF cell apoptosis and the anti-apoptotic effects of the caspase inhibitors were additive. Our data suggest that endoplasmic reticulum stress, likely mediated by NO, contributes to the AF cell apoptosis induced by cyclic stretch in addition to the mitochondrial pathway. These findings could be helpful to understand the mechanism of disc cell apoptosis, the root cause of IVD degeneration.     

Accelerated premature stress-induced senescence of young annulus fibrosus cells of rats by high glucose-induced oxidative stress

Purposes

Diabetes mellitus (DM) is thought to be an important aetiological factor in intervertebral disc degeneration. A glucose-mediated increase of oxidative stress is a major causative factor in development of diseases associated with DM. The aim of this study was to investigate the effect of high glucose on mitochondrial damage, oxidative stress and senescence of young annulus fibrosus (AF) cells.

Methods

AF cells were isolated from four-week-old young rats, cultured, and placed in either 10 % FBS (normal control) or 10 % FBS plus two different high glucose concentrations (0.1 M and 0.2 M) (experimental conditions) for one and three days. We identified and quantified the mitochondrial damage and reactive oxygen species (ROS) (oxidative stress). We also identified and quantified the occurrence of senescence and telomerase activity. Finally, the expressions of proteins were determined related to replicative senescence (p53-p21-pRB) and stress-induced senescence (p16-pRB).

Results

Two high glucoses enhanced the mitochondrial damage in young rat AF cells, which resulted in an excessive generation of ROS in a dose- and time-dependent manner for one and three days compared to normal control. Two high glucose concentrations increased the occurrence of senescence of young AF cells in a dose- and time-dependent manner. Telomerase activity declined in a dose- and time-dependent manner. Both high glucose treatments increased the expressions of p16 and pRB proteins in young rat AF cells for one and three days. However, compared to normal control, the expressions of p53 and p21 proteins were decreased in young rat AF cells treated with both high glucoses for one and three days.

Conclusions

The present study demonstrated that high glucose-induced oxidative stress accelerates premature stress-induced senescence in young rat AF cells in a dose- and time-dependent manner rather than replicative senescence. These results suggest that prevention of excessive generation of oxidative stress by strict blood glucose control could be important to prevent or to delay premature intervertebral disc degeneration in young patients with DM.     

Relationship between solute transport properties and tissue morphology in human annulus fibrosus

Poor nutritional supply to the intervertebral disc is believed to be an important factor leading to disc degeneration. However, little is known regarding nutritional transport in human annulus fibrosus (AF) and its relation to tissue morphology. We hypothesized that solute diffusivity in human AF is anisotropic and inhomogeneous, and that transport behaviors are associated with tissue composition and structure. To test these hypotheses, we measured the direction‐dependent diffusivity of a fluorescent molecule (fluorescein, 332 Da) in three regions of AF using a fluorescence recovery after photobleaching (FRAP) technique, and associated transport results to the regional variation in water content and collagen architecture in the tissue. Diffusivity in AF was anisotropic, with higher values in the axial direction than in the radial direction for all regions investigated. The values of the diffusion coefficient ranged from 0.38 ± 0.25 × 10^?6 cm²/s (radial diffusivity in outer AF) to 2.68 ± 0.84 × 10^?6 cm²/s (axial diffusivity in inner AF). In both directions, diffusivity decreased moving from inner to outer AF. Tissue structure was investigated using both scanning electron microscopy and environmental scanning electron microscopy. A unique arrangement of microtubes was found in human AF. Furthermore, we also found that the density of these microtubes varied moving from inner to outer AF. A similar trend of regional variation was found for water content, with the highest value also measured in inner AF. Therefore, we concluded that a relationship exists among the anisotropic and inhomogeneous diffusion in human AF and the structure and composition of the tissue. © 2009 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 27:1625–1630, 2009     

白细胞介素-6抑制白细胞介素-1β诱导的兔纤维环细胞凋亡的研究

目的探讨白细胞介素(IL)-6对IL-1β在体外诱导兔纤维环细胞凋亡作用的影响。方法以10μg/L浓度的IL-1β诱导体外培养的原代兔纤维环细胞发生凋亡,然后分别加入10μg/ L和100μg/L的IL-6以影响纤维环细胞凋亡进程。对细胞行Annexin-V-PI染色和Caspase-9功能染色,采用流式细胞仪检测结果。结果IL-1β诱导下,凋亡纤维环细胞比例由(2.67±1.08)%上升至(20.37±1.57)%,而在两种浓度IL-6的干预下,细胞凋亡比例分别为(18.17±4.68)%和(9.42±1.27)%,与IL-1β诱导退变的纤维环细胞差异有统计学意义(P＜0.05)。而Caspase-9功能阳性细胞也由(19.40±0.98)%分别降至(15.13±1.45)%和(10.17±2.50)%(P＜0.05)。结论IL-6对IL-1β诱导的体外兔纤维环细胞凋亡有明显的抑制作用,这种抑制作用可能是通过抑制纤维环细胞内Caspase-9的激活实现的。对IL-6抑制作用的进一步研究,将有助于明确椎间盘细胞凋亡的调控途径,为椎间盘退变的治疗寻找新切入点。     

Recombinant osteogenic protein-1 upregulates extracellular matrix metabolism by rabbit annulus fibrosus and nucleus pulposus cells cultured in alginate beads.

This study was performed to determine if recombinant human osteogenic protein-1 (rhOP-1) is effective in promoting matrix synthesis and matrix formation by rabbit nucleus pulposus (NP) and annulus fibrosus (AF) cells cultured in alginate beads. The effects of culturing the cells in the presence of various concentrations of rhOP-1 were assessed by measuring changes in cell proliferation, proteoglycan (PG) and collagen synthesis and mRNA expression, and in the matrix contents of PG and collagen, as indicators of matrix accumulation. At high concentrations, rhOP-1 had a moderate mitogenic effect on both NP and AF cells. It also stimulated the synthesis of PG and collagen in a dose-dependent manner: this was associated with a corresponding increase in the expression of mRNA for aggrecan core protein and collagen type II. The stimulatory effect of rhOP-1 on PG synthesis was more pronounced than that on collagen synthesis. Continuous treatment with rhOP-1 led to an increase in the total DNA, PG and collagen contents in both NP and AF cultures. The results presented here provide evidence of the ability of rhOP-1 to stimulate the metabolism of both rabbit AF and NP cells cultured in alginate beads.     

Shear mechanical properties of human lumbar annulus fibrosus.

Function, failure, and remodeling of the intervertebral disc are all related to the stress and strain fields in the tissue and may be calculated by finite element models with accurate material properties, realistic geometry, and appropriate boundary conditions. There is no comprehensive study in the literature investigating the shear material properties of the annulus fibrosus. This study obtained shear material properties of the annulus fibrosus and tested the hypothesis that these properties are affected by the amplitude and frequency of shearing, applied compressive stress, and degenerative state of the tissue. Cylindrical specimens with an axial orientation from seven nondegenerated and six degenerated discs were tested in torsional shear under dynamic and static conditions. Frequency sweep experiments over a physiological range of frequencies (0.1-100 rad/sec) at a shear strain amplitude of 0.05 rad were performed under three different axial compressive stresses (17.5, 25, and 35 kPa). At the largest compressive stress, shear strain sweep experiments (strain amplitude range: 0.005-0.15 rad at a frequency of 5 rad/sec) and transient stress-relaxation tests (shear strain range: 0.02-0.15 rad) were performed. The annulus fibrosus material was less stiff and more dissipative at larger shear strain amplitudes, stiffer at higher frequencies of oscillation, and stiffer and less dissipative at larger axial compressive stresses. The dynamic shear modulus, /G*/, had values ranging from 100 to 400 kPa, depending on the experimental condition and degenerative level. The shear behavior was also predominantly elastic, with values for the tangent of the phase angle (tandelta) ranging from 0.1 to 0.7. The annulus material also became stiffer and more dissipative with degenerative grade; however, this was not statistically significant. The results indicated that nonlinearities, compression/shear coupling, intrinsic viscoelasticity, and, to a lesser degree, degeneration all affect the shear material behavior of the annulus fibrosus, with important implications for load-carriage mechanisms in the intervertebral disc. These material complexities should be considered when choosing material constants for finite element models.     

Mechanics of oriented electrospun nanofibrous scaffolds for annulus fibrosus tissue engineering.

Engineering a functional replacement for the annulus fibrosus (AF) of the intervertebral disc is contingent upon recapitulation of AF structure, composition, and mechanical properties. In this study, we propose a new paradigm for AF tissue engineering that focuses on the reconstitution of anatomic fiber architecture and uses constitutive modeling to evaluate construct function. A modified electrospinning technique was utilized to generate aligned nanofibrous polymer scaffolds for engineering the basic functional unit of the AF, a single lamella. Scaffolds were tested in uniaxial tension at multiple fiber orientations, demonstrating a nonlinear dependence of modulus on fiber angle that mimicked the nonlinearity and anisotropy of native AF. A homogenization model previously applied to native AF successfully described scaffold mechanical response, and parametric studies demonstrated that nonfibrillar matrix, along with fiber connectivity, are key contributors to tensile mechanics for engineered AF. We demonstrated that AF cells orient themselves along the aligned scaffolds and deposit matrix that contributes to construct mechanics under loading conditions relevant to the in vivo environment. The homogenization model was applied to cell-seeded constructs and provided quantitative measures for the evolution of matrix and interfibrillar interactions. Finally, the model demonstrated that at fiber angles of the AF (28 degrees -44 degrees ), engineered material behaved much like native tissue, suggesting that engineered constructs replicate the physiologic behavior of the single AF lamella. Constitutive modeling provides a powerful tool for analysis of engineered AF neo-tissue and native AF tissue alike, highlighting key mechanical design criteria for functional AF tissue engineering.