Micro-porous PLGA/ß-TCP/TPU scaffolds prepared by solvent-based 3D printing for bone tissue engineering purposes.

The 3D printing process of fused deposition modelling is an attractive fabrication approach to create tissue-engineered bone substitutes to regenerate large mandibular bone defects, but often lacks desired surface porosity for enhanced protein adsorption and cell adhesion. Solvent-based printing leads to the spontaneous formation of micropores on the scaffold's surface upon solvent removal, without the need for further post processing. Our aim is to create and characterize porous scaffolds using a new formulation composed of mechanically stable poly(lactic-co-glycol acid) and osteoconductive ß-tricalcium phosphate with and without the addition of elastic thermoplastic polyurethane prepared by solvent-based 3D-printing technique. Large-scale regenerative scaffolds can be 3D-printed with adequate fidelity and show porosity at multiple levels analysed via micro-computer tomography, scanning electron microscopy and N2 sorption. Superior mechanical properties compared to a commercially available calcium phosphate ink are demonstrated in compression and screw pull out tests. Biological assessments including cell activity assay and live-dead staining prove the scaffold's cytocompatibility. Osteoconductive properties are demonstrated by performing an osteogenic differentiation assay with primary human bone marrow mesenchymal stromal cells. We propose a versatile fabrication process to create porous 3D-printed scaffolds with adequate mechanical stability and osteoconductivity, both important characteristics for segmental mandibular bone reconstruction.

A Novel Fiber-Reinforced Poroviscoelastic Bovine Intervertebral Disc Finite Element Model for Organ Culture Experiment Simulations.

Intervertebral disc (IVD) degeneration and methods for repair and regeneration have commonly been studied in organ cultures with animal IVDs under compressive loading. With the recent establishment of a novel multi-axial organ culture system, accurate predictions of the global and local mechanical response of the IVD are needed for control system development and to aid in experiment planning. This study aimed to establish a finite element model of bovine IVD capable of predicting IVD behavior at physiological and detrimental load levels. A finite element model was created based on the dimensions and shape of a typical bovine IVD used in the organ culture. The nucleus pulposus (NP) was modeled as a neo-Hookean poroelastic material and the annulus fibrosus (AF) as a fiber-reinforced poroviscoelastic material. The AF consisted of 10 lamella layers and the material properties were distributed in the radial direction. The model outcome was compared to a bovine IVD in a compressive stress-relaxation experiment. A parametric study was conducted to investigate the effect of different material parameters on the overall IVD response. The model was able to capture the equilibrium response and the relaxation response at physiological and higher strain levels. Permeability and elastic stiffness of the AF fiber network affected the overall response most prominently. The established model can be used to evaluate the response of the bovine IVD at strain levels typical for organ culture experiments, to define relevant boundaries for such studies, and to aid in the development and use of new multi-axial organ culture systems.

Physiological and degenerative loading of bovine intervertebral disc in a bioreactor: A finite element study of complex motions.

Intervertebral disc (IVD) degeneration and regenerative therapies are commonly studied in organ-culture experiments with uniaxial compressive loading. Recently, in our laboratory, we established a bioreactor system capable of applying loads in six degrees-of-freedom (DOF) to bovine IVDs, which replicates more closely the complex multi-axial loading of the IVD in vivo. However, the magnitudes of loading that are physiological (able to maintain cell viability) or mechanically degenerative are unknown for load cases combining several DOFs. This study aimed to establish physiological and degenerative levels of maximum principal strains and stresses in the bovine IVD tissue and to investigate how they are achieved under complex load cases related to common daily activities. The physiological and degenerative levels of maximum principal strains and stresses were determined via finite element (FE) analysis of bovine IVD subjected to experimentally established physiological and degenerative compressive loading protocols. Then, complex load cases, such as a combination of compression + flexion + torsion, were applied on the FE-model with increasing magnitudes of loading to discover when physiological and degenerative tissue strains and stresses were reached. When applying 0.1 MPa of compression and ±2-3° of flexion and ±1-2° of torsion the investigated mechanical parameters remained at physiological levels, but with ±6-8° of flexion in combination with ±2-4° of torsion, the stresses in the outer annulus fibrosus (OAF) exceeded degenerative levels. In the case of compression + flexion + torsion, the mechanical degeneration likely initiates at the OAF when loading magnitudes are high enough. The physiological and degenerative magnitudes can be used as guidelines for bioreactor experiments with bovine IVDs.

Adaptation of Fibril-Reinforced Poroviscoelastic Properties in Rabbit Collateral Ligaments 8 Weeks After Anterior Cruciate Ligament Transection.

Ligaments of the knee provide stability and prevent excessive motions of the joint. Rupture of the anterior cruciate ligament (ACL), a common sports injury, results in an altered loading environment for other tissues in the joint, likely leading to their mechanical adaptation. In the collateral ligaments, the patterns and mechanisms of biomechanical adaptation following ACL transection (ACLT) remain unknown. We aimed to characterize the adaptation of elastic and viscoelastic properties of the lateral and medial collateral ligaments eight weeks after ACLT. Unilateral ACLT was performed in six rabbits, and collateral ligaments were harvested from transected and contralateral knee joints after eight weeks, and from an intact control group (eight knees from four animals). The cross-sectional areas were measured with micro-computed tomography. Stepwise tensile stress-relaxation testing was conducted up to 6% final strain, and the elastic and viscoelastic properties were characterized with a fibril-reinforced poroviscoelastic material model. We found that the cross-sectional area of the collateral ligaments in the ACL transected knees increased, the nonlinear elastic collagen network modulus of the LCL decreased, and the amount of fast relaxation in the MCL decreased. Our results indicate that rupture of the ACL leads to an early adaptation of the elastic and viscoelastic properties of the collagen fibrillar network in the collateral ligaments. These adaptations may be important to consider when evaluating whole knee joint mechanics after ACL rupture, and the results aid in understanding the consequences of ACL rupture on other tissues.

Toward the Next Generation of Spine Bioreactors: Validation of an Ex Vivo Intervertebral Disc Organ Model and Customized Specimen Holder for Multiaxial Loading.

A new generation of bioreactors with integrated six degrees of freedom (6 DOF) aims to mimic more accurately the natural intervertebral disc (IVD) load. We developed and validated in a biological and mechanical study a specimen holder and corresponding ex vivo IVD organ model according to the bioreactor requirements for multiaxial loading and a long-term IVD culture. IVD height changes and cell viability were compared between the 6 DOF model and the standard 1 DOF model throughout the 3 weeks of cyclic compressive loading in the uniaxial bioreactor. Furthermore, the 6 DOF model and holder were loaded for 9 days in the multiaxial bioreactor under development using the same conditions, and the IVDs were evaluated for cell viability. The interface of the IVD model and specimen holder, enhanced with fixation screws onto the bone, was tested in compression, torsion, lateral bending, and tension. Additionally, critical motions such as tension and bending were assessed for a combination of side screws and top screws or side screws and adhesive. The 6 DOF model loaded in the uniaxial bioreactor maintained similar cell viability in the IVD regions as the 1 DOF model. The viability was high after 2 weeks throughout the whole IVD and reduced by more than 30% in the inner annulus fibrous after 3 weeks. Similarly, the IVDs remained highly viabile when cultured in the multiaxial bioreactor. In both models, IVD height changes after loading were in the range of typical physiological conditions. When differently directed motions were applied, the holder-IVD interface remained stable under hyper-physiological loading levels using a side screw approach in compression and torsion and the combination of side and top screws in tension and bending. We thus conclude that the developed holding system is mechanically reliable and biologically compatible for application in a new generation of multiaxial bioreactors.

Assessment of Ligament Viscoelastic Properties Using Raman Spectroscopy.

Injuries to the ligaments of the knee commonly impact vulnerable and physically active individuals. These injuries can lead to the development of degenerative diseases such as post-traumatic osteoarthritis (PTOA). Non-invasive optical modalities, such as infrared and Raman spectroscopy, provide means for quantitative evaluation of knee joint tissues and have been proposed as potential quantitative diagnostic tools for arthroscopy. In this study, we evaluate Raman spectroscopy as a viable tool for estimating functional properties of collateral ligaments. Artificial trauma was induced by anterior cruciate ligament transection (ACLT) in the left or right knee joint of skeletally mature New Zealand rabbits. The corresponding contralateral (CL) samples were extracted from healthy unoperated joints along with a separate group of control (CNTRL) animals. The rabbits were sacrificed at 8 weeks after ACLT. The ligaments were then harvested and measured using Raman spectroscopy. A uniaxial tensile stress-relaxation testing protocol was adopted for determining several biomechanical properties of the samples. Partial least squares (PLS) regression models were then employed to correlate the spectral data with the biomechanical properties. Results show that the capacity of Raman spectroscopy for estimating the biomechanical properties of the ligament samples varies depending on the target property, with prediction error ranging from 15.78% for tissue cross-sectional area to 30.39% for stiffness. The hysteresis under cyclic loading at 2 Hz (RMSE = 6.22%, Normalized RMSE = 22.24%) can be accurately estimated from the Raman data which describes the viscous damping properties of the tissue. We conclude that Raman spectroscopy has the potential for non-destructively estimating ligament biomechanical properties in health and disease, thus enhancing the diagnostic value of optical arthroscopic evaluations of ligament integrity.

Near infrared spectroscopic evaluation of biochemical and crimp properties of knee joint ligaments and patellar tendon.

Knee ligaments and tendons play an important role in stabilizing and controlling the motions of the knee. Injuries to the ligaments can lead to abnormal mechanical loading of the other supporting tissues (e.g., cartilage and meniscus) and even osteoarthritis. While the condition of knee ligaments can be examined during arthroscopic repair procedures, the arthroscopic evaluation suffers from subjectivity and poor repeatability. Near infrared spectroscopy (NIRS) is capable of non-destructively quantifying the composition and structure of collagen-rich connective tissues, such as articular cartilage and meniscus. Despite the similarities, NIRS-based evaluation of ligament composition has not been previously attempted. In this study, ligaments and patellar tendon of ten bovine stifle joints were measured with NIRS, followed by chemical and histological reference analysis. The relationship between the reference properties of the tissue and NIR spectra was investigated using partial least squares regression. NIRS was found to be sensitive towards the water (R2CV = .65) and collagen (R2CV = .57) contents, while elastin, proteoglycans, and the internal crimp structure remained undetectable. As collagen largely determines the mechanical response of ligaments, we conclude that NIRS demonstrates potential for quantitative evaluation of knee ligaments.

Biomechanical, biochemical, and near infrared spectral data of bovine knee ligaments and patellar tendon.

Knee joint ligaments and patellar tendon are rope-like tissues that enable the proper function of the knee by connecting the bones that form the joint. A better understanding of ligament structure-function relationships is needed to develop objective and reliable diagnostic methods for ligaments. Recently, arthroscopic near infrared spectroscopy (NIR) has shown the potential to quantitatively evaluate the health of the cartilages and menisci of the knee. In this dataset, we present a unique combination of NIR spectral data, biomechanical properties, and biochemical composition of bovine primary knee ligaments and patellar tendon (10 knees, 50 tissue samples). NIR spectral data were measured at 5 locations in each sample, biomechanical properties were obtained with tensile testing, and biochemical composition was quantified using colorimetric biochemical methods. The data can be reused for investigations of structure-function relationships of knee ligaments and patellar tendon, for the development of NIR spectroscopic methods to quantify the health of these tissues, and to develop new computational models to describe ligament and tendon biomechanics.

Structure, composition and fibril-reinforced poroviscoelastic properties of bovine knee ligaments and patellar tendon.

Tissue-level stress-relaxation of ligaments and tendons in the toe region is characterized by fast and long-term relaxations and an increase in relaxation magnitude with strain. Characterizing the compositional and structural origins of these phenomena helps in the understanding of mechanisms of ligament and tendon function and adaptation in health and disease. A three-step tensile stress-relaxation test was conducted on dumbbell-shaped pieces of bovine knee ligaments and patellar tendon (PT) (n = 10 knees). Their mechanical behaviour was characterized by a fibril-reinforced poroviscoelastic material model, able to describe characteristic times and magnitudes of fast and long-term relaxations. The crimp angle and length of tissues were measured with polarized light microscopy, while biochemical contents were determined by colorimetric biochemical methods. The long-term relaxation time was longer in the anterior cruciate ligament (ACL) and PT compared with collateral ligaments (p < 0.05). High hydroxyproline content predicted greater magnitude and shorter time of both fast and long-term relaxation. High uronic acid content predicted longer time of long-term relaxation, whereas high crimp angle predicted higher magnitude of long-term relaxation. ACL and PT are better long-term stabilizers than collateral ligaments. The long-term relaxation behaviour is affected or implied by proteoglycans and crimp angle, possibly relating to slow structural reorganization of the tissue.

The effect of different preconditioning protocols on repeatability of bovine ACL stress-relaxation response in tension.

Mechanical characterization of soft tissues such as ligaments remains challenging. There is variability in the measured material parameters of ligaments, most of which is related to natural tissue variability, but some of it can be a result of using different testing protocols. Generally preconditioning (cyclic loading-unloading) is performed prior to actual tests to reduce the experimental variability. Commonly, preconditioning protocols for ligaments with a small strain level and 10 sinusoidal loading-unloading cycles are used. The effect of preconditioning and its parameters including strain level, number of cycles and number of preconditioning repetitions on the repeatability of tensile stress-relaxation tests are poorly known for knee ligaments. In the present study, forty-eight dumbbell-shaped bovine anterior cruciate ligament (ACL) samples were used to evaluate the repeatability of stress-relaxation response. Different preconditioning protocols with 2% and 6% strain levels and 1, 5 or 10 preconditioning repetitions were applied. After preconditioning, one-step stress-relaxation test was carried out twice with an hour resting period in between the tests. The equilibrium stress showed no systematic bias when only one preconditioning repetition was applied (2.0 ±â€¯3.1% difference and p > 0.05 between repeated tests). Systematic bias in the peak-to-equilibrium stress ratio was not observed when higher strain level and number of repetitions were used (0.5 ±â€¯1.6% difference and p > 0.05 between repeated tests). In conclusion, the commonly used preconditioning protocol is capable of producing repeatable equilibrium stress levels of bovine ACLs from stress-relaxation tests in tension. However, if repeatable peak-to-equilibrium stress ratio is desirable, higher strain and number of preconditioning repetitions are recommended.

Near Infrared Spectroscopic Evaluation of Ligament and Tendon Biomechanical Properties.

Knee ligaments and tendons are collagen-rich viscoelastic connective tissues that provide vital mechanical stabilization and support to the knee joint. Deterioration of ligaments has an adverse effect on the health of the knee and can eventually lead to ligament rupture and osteoarthritis. In this study, the feasibility of near infrared spectroscopy (NIRS) was, for the first time, tested for evaluation of ligament and tendon mechanical properties by performing measurements on bovine stifle joint ligament (N = 40) and patellar tendon (N = 10) samples. The mechanical properties of the samples were determined using a uniaxial tensile testing protocol. Partial least squares regression models were then developed to determine if morphological, viscoelastic, and quasi-static properties of the samples could be predicted from the NIR spectra. Best performance of NIRS in predicting mechanical properties was observed for toughness at yield point (median [Formula: see text], median normalized [Formula: see text]), toughness at failure point (median [Formula: see text], median normalized [Formula: see text]), and the ultimate strength of the ligament/tendon (median [Formula: see text], median normalized [Formula: see text]). Thus, we show that NIRS is capable of estimating ligament and tendon biomechanical properties, especially in parameters related to tissue failure. We believe this method could substantially enhance the currently limited arthroscopic evaluation of ligaments and tendons.