Biomimetic De Novo Ligament Regeneration

Ligament injuries affect millions of people around the world every year which can happen from various reasons from sports, accidents, or simple daily activities and they present a challenging problem because of the poor regenerative capacity of ligament tissue. Even with advances in medical technology, current treatment options, including biological grafts such as autografts and allografts or synthetic ligaments remain inadequate for long-term recovery. The primary complications include poor biological integration, immune rejection, and limited mechanical durability, all of which contribute to high rates of graft failure, fatigue, and re-rupture. The use of biological grafts, such as autografts harvested from the patient’s own tissue presents issues like donor site morbidity and extended recovery times. Allografts, on the other hand, carry the risk of immune rejection and infection as well as inconsistent healing outcomes. On the other hand, synthetic ligaments including materials made from polyethylene terephthalate may offer some mechanical benefits but they still fail in the long term due to their lack of biological integration, however, they are also susceptible to wear, fatigue, and even graft re-rupture due to the inability of synthetic materials to mimic the complex structural and biochemical environment of native ligaments. Therefore, there is an urgent need for innovative solutions that can better replicate the mechanical function of ligaments as well as promote tissue regeneration and biological integration. To this account, recent paper published in Advanced Materials Journal and conducted by Yu-Chung Liu, Wei-Yuan Huang, Hao-Xuan Chen, and led by Professor Tzu-Wei Wang from the National Tsing Hua University together with Dr. Shih-Heng Chen from the Chang Gung Memorial Hospital and Dr. Chen-Hsiang Kuan from the Taiwan University Hospital, the researchers developed an innovative synthetic ligament that closely mimics the natural structure and function of native ligaments.

The team used a technique known as interfacial polyelectrolyte complexation (IPC) to create fibers that can form a scaffold mimicking the hierarchical structure of native ligaments where they introduced a hydroxyapatite (HAp) mineral gradient at the ends of the scaffold and this replicated the natural transition between bone and ligament, known as the enthesis. Additionally, the authors integrated connective tissue growth factor (CTGF) and mesenchymal stem cells (MSCs) to further enhance the scaffold’s regenerative potential. To elaborate, they began their experiments by fabricating the scaffold using IPC spinning. This technique allowed the team to generate biocompatible fibers through electrostatic interactions between polycation and polyanion pairs, such as poly-D-lysine (PDL) and pectin. These fibers were assembled in a hierarchical fashion to mimic the fascicle and sub-fascicle structures found in natural ligaments. Using scanning electron microscopy, the researchers observed that the fibers displayed a porous, multi-layered architecture, with distinct primary and secondary fiber bundles that closely resembled the complex microstructure of ligaments and that confirmed to them that their novel fabrication method was successful in replicating the ligament’s complex architecture. Additionally, they further strengthen the scaffold with the incorporation of HAp mineral gradient at both ends of the structure. The HAp coating, deposited using a wet chemical synthesis method, was designed to mimic the enthesis, the natural transition zone between bone and ligament. This mineral gradient was expected to reduce stress concentration and improve integration with bone tissue. The researchers confirmed the successful creation of the gradient through advanced analytical tools including X-ray diffraction and energy-dispersive X-ray spectroscopy which demonstrated a consistent deposition of hydroxyapatite crystals and confirmed that the gradient not only improved the scaffold’s ability to integrate with bone but also enhanced its mechanical stability, thus addressing a critical issue with traditional synthetic ligaments that often fail at the bone-ligament junction.

Next the authors tested the mechanical properties of the scaffold under physiologically relevant conditions and conducted tensile tests to measure the scaffold’s ultimate tensile strength and found that cross-linking the IPC fibers using poly-D-lysine and pectin significantly increased their strength. The cross-linked fibers also showed improved stiffness and fatigue resistance which were critical for mimicking the durability of native ligaments under repetitive mechanical stress. Additionally, the introduction of a collagen coating further enhanced the scaffold’s viscoelastic properties and that allowed it to better withstand cyclic loading and stress relaxation, which are common mechanical demands in everyday movements. The researchers also focused on the scaffold’s ability to support tissue regeneration. They incorporated CTGF into the scaffold using a core-sheath structure to ensure a sustained release of the growth factor. In vitro experiments showed that the release of CTGF was well-controlled, with minimal burst release, allowing the growth factor to remain active for an extended period. Additionally, the team seeded MSCs onto the scaffold to promote healing at the cellular level and found that the scaffold did indeed support well MSC adhesion, proliferation, and differentiation with no cytotoxic effect indicating high safety. To further validate their biomedical engineering design, the team of experts implanted the scaffold in a rabbit model of anterior cruciate ligament reconstruction. Over 10 weeks, they assessed tissue integration and healing at the graft-to-bone interface where histological analysis showed significant tissue infiltration within the scaffold with enhanced collagen deposition and cellularity in the groups treated with CTGF and MSCs. By 10 weeks, the scaffold had become well-integrated with the surrounding tissue, and the groups treated with both CTGF and MSCs had the most pronounced healing, with a substantial increase in collagen content and ligament-specific protein markers like tenomodulin and tenascin C. According to the authors, these experimental findings suggested that the combined use of growth factors and stem cells improved the scaffold’s ability to promote ligament regeneration and healing at the bone-ligament interface.

The researchers also used micro-computed tomography to evaluate bone healing at the graft-bone interface and very high resolution. They observed that the scaffolds with the HAp gradient led to significantly higher bone mineral density and bone volume fraction compared to traditional grafts. This confirmed that the mineral gradient played a crucial role in promoting osseointegration and bone formation at the scaffold’s ends. In terms of mechanical performance, biomechanical testing showed that the scaffolds treated with CTGF and MSCs nearly matched the tensile strength of autografts, providing further evidence that the engineered scaffold could withstand the physical demands of ligament repair. In conclusion, Professor Tzu-Wei Wang and colleagues successfully developed a synthetic scaffold that mimics well the natural structure and function of ligaments and by this provided a long-waited solution to the major challenges faced in ligament repair and their medical treatment including poor graft integration, inadequate mechanical strength, and the risk of re-rupture. Moreover, we believe the authors’ introduction of a HAp mineral gradient at the ends of the scaffold is noteworthy as it closely replicates the natural bone-ligament interface and facilitate better integration and reduce stress concentrations that can lead to graft failure. The implications of Professor Tzu-Wei Wang and his team work are far-reaching because if these advancements are used in tissue engineering it potentially will improve patient outcomes who are undergoing orthopedic surgery with the expected advantages of faster healing times, better long-term outcomes, and reduced complications compared to current graft options, which often suffer from delayed healing or immune rejection. Additionally, the innovations of IPC spinning technique proposed can be used in other areas of tissue repair beyond ligaments to create hierarchical fiber structures could potentially be applied to other types of connective tissues, such as tendons or even more complex musculoskeletal systems.

In a statement to Advances in Engineering, Professor Tzu-Wei Wang  said: “This work introduces a physiologically-inspired strategy to devise a biomimetic ligament replacement that effectively emulate native ligament performance for facilitating ligament regeneration. The intricate hierarchical structure closely mirrors the fiber arrangement observed in native ligaments, providing guidance for regenerative process. The hydroxyapatite gradient distribution of mineralized constituents promotes the transition from soft to hard tissue and enhances the healing process at the ligament-to-bone interface. Custom-tailored viscoelastic properties are designed to mirror those inherent in native ligament, ensuring proper load-bearing capacity and stability. The introduction of stem cells and delivery of growth factors facilitate the formation of functional ligamentous tissue. Through these advancements, this research aims at improving clinical outcomes, facilitate patient recovery, and provide a long-lasting and effective solution for ligament injuries”