Jeffrey P. Spalazzi

Novel Nanofiber-Based Scaffold for Rotator Cuff Repair and Augmentation

The debilitating effects of rotator cuff tears and the high incidence of failure associated with current grafts underscore the clinical demand for functional solutions for tendon repair and augmentation. To address this challenge, we have designed a poly(lactide-co-glycolide) (PLGA) nanofiber-based scaffold for rotator cuff tendon tissue engineering. In addition to scaffold design and characterization, the objective of this study was to evaluate the attachment, alignment, gene expression, and matrix elaboration of human rotator cuff fibroblasts on aligned and unaligned PLGA nanofiber scaffolds. Additionally, the effects of in vitro culture on scaffold mechanical properties were determined over time. It has been hypothesized that nanofiber organization regulates cellular response and scaffold properties. It was observed that rotator cuff fibroblasts cultured on the aligned scaffolds attached along the nanofiber long axis, whereas the cells on the unaligned scaffold were polygonal and randomly oriented. Moreover, distinct integrin expression profiles on these two substrates were observed. Quantitative analysis revealed that cell alignment, distribution, and matrix deposition conformed to nanofiber organization and that the observed differences were maintained over time. Mechanical properties of the aligned nanofiber scaffolds were significantly higher than those of the unaligned, and although the scaffolds degraded in vitro, physiologically relevant mechanical properties were maintained. These observations demonstrate the potential of the PLGA nanofiber-based scaffold system for functional rotator cuff repair. Moreover, nanofiber organization has a profound effect on cellular response and matrix properties, and it is a critical parameter for scaffold design.

In vivo evaluation of a multiphased scaffold designed for orthopaedic interface tissue engineering and soft tissue-to-bone integration

Achieving functional graft integration with subchondral bone poses a significant challenge for orthopaedic soft tissue repair and reconstruction. Soft tissues such as the anterior cruciate ligament (ACL) integrate with bone through a fibrocartilage interface, which minimizes stress concentrations and mediates load transfer between soft and hard tissues. We propose that biological fixation can be achieved by regenerating this fibrocartilage interface on biological or synthetic ACL grafts. This study focuses on the in vivo evaluation of a stratified scaffold predesigned to mimic the multitissue transition found at the ACL-to-bone interface. Specifically, the scaffold consists of three distinct yet continuous phases: Phase A for ligament formation, Phase B for the interface, and Phase C for the bone region. Interface-relevant cell types, specifically fibroblasts, chondrocytes, and osteoblasts, will be tri-cultured on this scaffold, and the formation of cell type- and phase-specific matrix heterogeneity as well as fibrocartilage formation will be evaluated over 8 weeks in a subcutaneous athymic rat model. Acellular scaffolds as well as scaffolds co-cultured with fibroblasts and osteoblasts will serve as controls. It was found that the triphasic scaffold supported multilineage cellular interactions as well as tissue infiltration and abundant matrix production in vivo. In addition, controlled phase-specific matrix heterogeneity was induced on the scaffold, with distinct mineral and fibrocartilage-like tissue regions formed in the tri-cultured group. Cell seeding had a positive effect on both host infiltration and matrix elaboration, which also translated into increased mechanical properties in the seeded groups compared to the acellular controls. In summary, the biomimetic and multiphasic design coupled with spatial control of cell distribution enables multitissue regeneration on the stratified scaffold, and demonstrates the potential for regenerating the interface between soft tissue grafts and bone.

Osteoblast and chondrocyte interactions during coculture on scaffolds

Examining matrix and substrate-dependent effects on the formation of functional bone-cartilage interfaces.

Quantitative mapping of matrix content and distribution across the ligament-to-bone insertion.

The interface between bone and connective tissues such as the Anterior Cruciate Ligament (ACL) constitutes a complex transition traversing multiple tissue regions, including non-calcified and calcified fibrocartilage, which integrates and enables load transfer between otherwise structurally and functionally distinct tissue types. The objective of this study was to investigate region-dependent changes in collagen, proteoglycan and mineral distribution, as well as collagen orientation, across the ligament-to-bone insertion site using Fourier transform infrared spectroscopic imaging (FTIR-I). Insertion site-related differences in matrix content were also evaluated by comparing tibial and femoral entheses. Both region- and site-related changes were observed. Collagen content was higher in the ligament and bone regions, while decreasing across the fibrocartilage interface. Moreover, interfacial collagen fibrils were aligned parallel to the ligament-bone interface near the ligament region, assuming a more random orientation through the bulk of the interface. Proteoglycan content was uniform on average across the insertion, while its distribution was relatively less variable at the tibial compared to the femoral insertion. Mineral was only detected in the calcified interface region, and its content increased exponentially across the mineralized fibrocartilage region toward bone. In addition to new insights into matrix composition and organization across the complex multi-tissue junction, findings from this study provide critical benchmarks for the regeneration of soft tissue-to-bone interfaces and integrative soft tissue repair.

FTIR-I compositional mapping of the cartilage-to-bone interface as a function of tissue region and age.

Soft tissue‐to‐bone transitions, such as the osteochondral interface, are complex junctions that connect multiple tissue types and are critical for musculoskeletal function. The osteochondral interface enables pressurization of articular cartilage, facilitates load transfer between cartilage and bone, and serves as a barrier between these two distinct tissues. Presently, there is a lack of quantitative understanding of the matrix and mineral distribution across this multitissue transition. Moreover, age‐related changes at the interface with the onset of skeletal maturity are also not well understood. Therefore, the objective of this study is to characterize the cartilage‐to‐bone transition as a function of age, using Fourier transform infrared spectroscopic imaging (FTIR‐I) analysis to map region‐dependent changes in collagen, proteoglycan, and mineral distribution, as well as collagen organization. Both tissue‐dependent and age‐related changes were observed, underscoring the role of postnatal physiological loading in matrix remodeling. It was observed that the relative collagen content increased continuously from cartilage to bone, whereas proteoglycan peaked within the deep zone of cartilage. With age, collagen content across the interface increased, accompanied by a higher degree of collagen alignment in both the surface and deep zone cartilage. Interestingly, regardless of age, mineral content increased exponentially across the calcified cartilage interface. These observations reveal new insights into both region‐ and age‐dependent changes across the cartilage‐to‐bone junction and will serve as critical benchmark parameters for current efforts in integrative cartilage repair.

Biomimetic Stratified Scaffold Design for Ligament-to-Bone Interface Tissue Engineering

The emphasis in the field of orthopaedic tissue engineering is on imparting biomimetic functionality to tissue engineered bone or soft tissue grafts and enabling their translation to the clinic. A significant challenge in achieving extended graft functionality is engineering the biological fixation of these grafts with each other as well as with the host environment. Biological fixation will require re-establishment of the structure-function relationship inherent at the native soft tissue-to-bone interface on these tissue engineered grafts. To this end, strategic biomimicry must be incorporated into advanced scaffold design. To facilitate integration between distinct tissue types (e.g., bone with soft tissues such as cartilage, ligament, or tendon), a stratified or multi-phasic scaffold with distinct yet continuous tissue regions is required to pre-engineer the interface between bone and soft tissues. Using the ACL-to-bone interface as a model system, this review outlines the strategies for stratified scaffold design for interface tissue engineering, focusing on identifying the relevant design parameters derived from an understanding of the structure-function relationship inherent at the soft-to-hard tissue interface. The design approach centers on first addressing the challenge of soft tissue-to-bone integration ex vivo, and then subsequently focusing on the relatively less difficult task of bone-to-bone integration in vivo. In addition, we will review stratified scaffold design aimed at exercising spatial control over heterotypic cellular interactions, which are critical for facilitating the formation and maintenance of distinct yet continuous multi-tissue regions. Finally, potential challenges and future directions in this emerging area of advanced scaffold design will be discussed.

Elastographic Imaging of the Strain Distribution at the Anterior Cruciate Ligament and ACL-Bone Insertions

The anterior cruciate ligament (ACL) functions as a mechanical stabilizer in the tibiofemoral joint. Over 250,000 Americans each year suffer ACL ruptures and tears, making the ACL the most commonly injured knee ligament. Methods which permit the in situ monitoring of changes in ACL graft mechanical properties during healing are needed. A long term goal in ACL reconstruction is to regenerate the ACL-bone interface. To this end, an understanding of mechanical properties of the ligament-bone interface is needed. However, experimental determination has been difficult due the small length scale (<1 mm) involved and limited resolution of standard techniques. The current study uses elastography to characterize the functional properties of the ACL and the ACL-bone interface under applied load. In a first experiment, bovine joints were excised, cast in an agar gel matrix and externally compressed. In a second experiment, tibiofemoral joints were mounted on a MTS 858 Bionix Testing System. The ACL was loaded at different strain rates and tested to failure while RF data was collected at 5 MHz. For both tensile and compression testing, axial elastograms between successive RF frames were generated using cross-correlation and recorrelation techniques. When the ACL-bone complex was tested in the tibial alignment on the MTS system, compressive strains were found to dominate at the tibial insertion. Compressive strains were observed in the ligament proper when the transducer beam was aligned with respect to the insertion during loading. The distribution of tensile and compressive strain varied as a function of strain rate during testing and between loading and unloading. These preliminary results agree with those of prior FEA model predictions. In addition, a narrow band of high strain in the middle of the ACL was detected during compression that is considered to be a softer region of the ACL containing a highly collagenous structure. These preliminary results on ACL geometry and function indicate that elastography can provide important information in understanding the structure and function of both the ACL and the ACL-bone insertion. Ongoing studies focus on in-depth evaluation of the mechanical properties existing at the ACL and ACL-bone insertions

Stratified scaffold design for engineering composite tissues.

A significant challenge to orthopaedic soft tissue repair is the biological fixation of autologous or allogeneic grafts with bone, whereby the lack of functional integration between such grafts and host bone has limited the clinical success of anterior cruciate ligament (ACL) and other common soft tissue-based reconstructive grafts. The inability of current surgical reconstruction to restore the native fibrocartilaginous insertion between the ACL and the femur or tibia, which minimizes stress concentration and facilitates load transfer between the soft and hard tissues, compromises the long-term clinical functionality of these grafts. To enable integration, a stratified scaffold design that mimics the multiple tissue regions of the ACL interface (ligament-fibrocartilage-bone) represents a promising strategy for composite tissue formation. Moreover, distinct cellular organization and phase-specific matrix heterogeneity achieved through co- or tri-culture within the scaffold system can promote biomimetic multi-tissue regeneration. Here, we describe the methods for fabricating a tri-phasic scaffold intended for ligament-bone integration, as well as the tri-culture of fibroblasts, chondrocytes, and osteoblasts on the stratified scaffold for the formation of structurally contiguous and compositionally distinct regions of ligament, fibrocartilage and bone. The primary advantage of the tri-phasic scaffold is the recapitulation of the multi-tissue organization across the native interface through the layered design. Moreover, in addition to ease of fabrication, each scaffold phase is similar in polymer composition and therefore can be joined together by sintering, enabling the seamless integration of each region and avoiding delamination between scaffold layers.

Elastographic imaging of strain distribution within the anterior cruciate ligament and at the acl-bone insertions

The anterior cruciate ligament (ACL) functions as a mechanical stabilizer in the tibiofemoral joint. Over 250,000 Americans each year suffer from ACL ruptures and tears, making the ACL the most commonly injured knee ligament. A long term goal of our research program is to promote graft-bone integration via the regeneration of the native ligament-bone interface. To this end, we have focused on the design of biomimetic scaffolds combined with tissue engineering to induce organized interface regeneration. An understanding of mechanical properties of the ligament-bone interface is critical for biomimetic scaffold design and clinical evaluation. To date, experimental determination has been difficult due to the small length scale (< 1 mm) involved at the ACL insertions. This study utilizes ultrasound elastography to characterize the functional properties of the ACL and the ACL-bone interface under applied loading. Specifically, the tibiofemoral joints were mounted on a biomechanics material testing system and loaded in tension while RF data was collected. Axial elastograms between successive RF frames were generated using cross-correlation and recorrelation techniques. Elastography analyses revealed that when the joint is in tension, complex strains with both compressive and tensile components were found at the tibial insertion. These results are in agreement with those of prior finite element analysis (FEA) model predictions. In addition, the magnitude of displacement was found to be the highest at the ACL proper and decreased in value from ligament to bone. Our results indicate that elastography is a novel and useful method in understanding the mechanical properties of the ligament itself and the ligament-bone interface.

Innovative Scaffold Design for Soft Tissue-to-Bone Interface Tissue Engineering

Soft tissue-based ACL reconstruction grafts are limited by their inability to reestablish a functional interface with bone tissue[1–2]. The native ACL-bone interface consists of three regions: ligament, fibrocartilage, and bone[3–5]. Graft integration is a critical factor governing its clinical success, and the regeneration of an anatomic interface on synthetic or biological ACL grafts will improve clinical outcome. Our interface tissue engineering effort has focused on biomimetic scaffold design to recapitulate the inherent complexity of the ligament-to-bone interface and ultimately, to guide interface regeneration. To this end, we have designed a tri-phasic scaffold comprised of three distinct yet continuous phases, each designed for the formation of a specific tissue type found at the ACL-to-bone interface, as well as a bi-phasic collar to promote the formation of fibrocartilage on ACL reconstruction grafts and also enhance osteointegration.Copyright