Anterior Cruciate Ligament Regeneration Research Articles

Braided biomimetic PCL grafts for anterior cruciate ligament repair and regeneration

Anterior cruciate ligament (ACL) is a knee joint stabilizer with a limited regeneration capacity mainly because of low cellular content. State-of-the-art procedures are unable to restore the functions of the tissue as demonstrated by limited success rates. Regenerative engineering can offer a solution for restoring the functions of torn/ruptured ligaments provided that biomimetic grafts are available as grafts/scaffolds. However, a model construct to test behavior of cells to better understand the healing mechanism of ACL is still missing. This study, firstly, aimed at creating an injured rabbit ACL model. Then, the injured and healthy ACL tissues were characterized in terms of alignment and diameter distributions of collagen fibrils. Next, polycaprolactone (PCL) grafts were prepared from braided electrospun meshes and were characterized in terms of alignment and diameter distributions of fibers. Finally, biomechanical properties of ACL tissue and mechanical properties of PCL grafts were determined and compared. Findings demonstrated that distributions of the fiber diameters of PCL electrospun grafts were similar to diameter distribution of collagens of healthy and injured rabbit ACL. The novelty of this study relies on the determination of the diameter distribution of collagens of healthy and injured rabbit ACL tissues, and fabrication of PCL grafts with diameter distributions similar to that seen in healthy and injured ACLs. This study is significant because it addresses a worldwide clinical problem associated with millions of patients. The fibrous biomimetic graft designed in this study is different from the traditional grafts that exhibit unimodal distribution, and it is expected to have a significant contribution to ACL regeneration efforts.

FABRICATION OF BIOACTIVE COLLAGEN ROPE FOR ANTERIOR CRUCIATE LIGAMENT RECONSTRUCTION

To address the current challenge of anterior cruciate ligament (ACL) reconstruction, this study is the first to fabricate a braided collagen rope (BCR) which mimics native hamstring for ACL reconstruction. The study aims to evaluate the biological and biomechanical properties of BCR both in vivo and vitro.Rabbit ACL reconstruction model using collagen rope and autograft (hamstring tendon) was conducted. The histological and biomechanical evaluations were conducted at 6-, 12-, 18, 26-week post-operation. In vitro study included cell morphology analysis, cell function evaluation and RNA sequencing of the tenocytes cultured on BCR. A cadaver study was also conducted to verify the feasibility of BCR for ACL reconstruction.BCR displays satisfactory mechanical strength similar to hamstring graft for ACL reconstruction in rabbit. Histological assessment showed BCR restore ACL morphology at 26 weeks similar to native ACL. The superior dynamic ligamentization in BCR over autograft group was evidenced by assessment of cell and collagen morphology and orientation. The in vitro study showed that the natural collagen fibres within BCR enables to signal the morphology adaptation and orientation of human tenocytes in bioreactor. BCR enables to enhance cell proliferation and tenogenic expression of tenocytes as compared to hydrolysed collagen. We performed an RNA-Sequencing (RNA-seq) experiment where RNA was extracted from tenocyte seeded with BCR. Analysis of enriched pathways of the up-regulated genes revealed that the most enriched pathways were the Hypoxia-inducible factor 1-alpha (HIF1A) regulated networks, implicating the possible mechanism BCR induced ACL regeneration. The subsequent cadaver study was conducted to proof the feasibility of BCR for ACL reconstruction.This study demonstrated the proof-of-concept of bio-textile braided collagen rope for ACL reconstruction, and the mechanism by which BCR induces natural collagen fibres that positively regulate morphology and function of tenocytes.

Collagen Fibril Diameter Distribution of Sheep Anterior Cruciate Ligament.

The anterior cruciate ligament (ACL) tissue is a soft tissue connecting the femur and tibia at the knee joint and demonstrates a limited capacity for self-regeneration due to its low vascularity. The currently available clinical procedures are unable to fully restore damaged ACL tissue, and tissue engineering can offer options with a potential of restoring the torn/ruptured ACL by using biomimetic constructs that are similar to native tissue in terms of structure, composition, and functions. However, a model substrate to understand how the ACL cells regenerate the injured tissue is still not available. In this study, it is hypothesized that the nanofiber-based model substrate with bimodal and unimodal fiber diameter distributions will mimic the diameter distribution of collagen fibrils seen in healthy and injured sheep ACL, respectively. The aims were to (i) create an ACL injury in a sheep ACL by applying extensional force to rupture the healthy ACL tissue, (ii) measure the collagen fibril diameter distributions of healthy and injured ACL, (iii) fabricate polycaprolactone (PCL) nanofiber-based model constructs using electrospinning with diameter distributions similar to healthy and injured ACL tissue, and (iv) measure mechanical properties of ACL tissue and PCL electrospun constructs. The results showed that the fiber diameter distributions of PCL electrospun constructs and those of the healthy and injured ACL tissues were similar. The novelty in this investigation is that the collagen fibril diameter distribution of healthy and injured sheep ACL tissues was reported for the first time. The study is significant because it aims to create a model construct to solve an important orthopedic-related clinical problem affecting millions of people globally. The model construct fabricated in this work is expected to have an important impact on ACL regeneration efforts.

Electrospun poly(3-hydroxybutyrate-co-3-hydroxyvalerate) scaffolds – a step towards ligament repair applications

ABSTRACT The incidence of anterior cruciate ligament (ACL) ruptures is approximately 50 per 100,000 people. ACL rupture repair methods that offer better biomechanics have the potential to reduce long term osteoarthritis. To improve ACL regeneration biomechanically similar, biocompatible and biodegradable tissue scaffolds are required. Poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV), with high 3-hydroxyvalerate (3HV) content, based scaffold materials have been developed, with the advantages of traditional tissue engineering scaffolds combined with attractive mechanical properties, e.g., elasticity and biodegradability. PHBV with 3HV fractions of 0 to 100 mol% were produced in a controlled manner allowing specific compositions to be targeted, giving control over material properties. In conjunction electrospinning conditions were altered, to manipulate the degree of fibre alignment, with increasing collector rotating speed used to obtain random and aligned PHBV fibres. The PHBV based materials produced were characterised, with mechanical properties, thermal properties and surface morphology being studied. An electrospun PHBV fibre mat with 50 mol% 3HV content shows a significant increase in elasticity compared to those with lower 3HV content and could be fabricated into aligned fibres. Biocompatibility testing with L929 fibroblasts demonstrates good cell viability, with the aligned fibre network promoting fibroblast alignment in the axial fibre direction, desirable for ACL repair applications. Dynamic load testing shows that the 50 mol% 3HV PHBV material produced can withstand cyclic loading with reasonable resilience. Electrospun PHBV can be produced with low batch variability and tailored, application specific properties, giving these biomaterials promise in tissue scaffold applications where aligned fibre networks are desired, such as ACL regeneration.

A Pilot Study of Aptamer-Conjugated Silk Ligament with MSCs Recruitment Ability for ACL Reconstruction

ABSTRACT Anterior cruciate ligament (ACL) rupture is one of the most common knee injuries. Due to the limited self-healing capacity, complex structure, and mechanical environment, the regeneration of ACL remains a challenge. To achieve fast recovery and long-term ACL regeneration, biodegradable scaffolds with excellent initial mechanical support and cell recruitment ability have gained increasing research interest. The aptamer-functionalized silk fiber was fabricated by embedding silk fiber into morpholinoethanesulfonic/1-ethyl-3-[3-dimethylaminopropyl] carbodiimide hydrochloride, N-hydroxysuccinimide buffer with aptamer 19s (Apt19s) and characterized in vitro. The clinically size-matched silk ligament was braided utilizing commercial methodology (polyethylene terephthalate ligament), and the tensile strength was analyzed. A total of 18 rabbits were used to test the early mesenchymal stem cell (MSC) recruitment of Apt19s-functionalized silk ligaments. Macroscopic analysis and hematoxylin and eosin and immunohistochemistry stainings were applied at 3, 5, and 7 days post-implantation. The ultimate strength of silk fiber was lower than that of PET fibers. However, the prepared silk ligament had comparable mechanical strength to the native ACL. In vitro, the Apt19s-functionalized fiber recruited bone marrow-derived MSCs. In vivo, Apt19s-functionalized silk ligament enhanced early MSCs recruitment after implantation in comparison with the aptamer-free ligament. The Apt19s-functionalized scaffold silk ligament exhibited comparable mechanical strength with native ACL and enhanced endogenous MSC homing.

Mechano Growth Factor Accelerates ACL Repair and Improves Cell Mobility of Mechanically Injured Human ACL Fibroblasts by Targeting Rac1-PAK1/2 and RhoA-ROCK1 Pathways

Exceeded mechanical stress leads to a sublethal injury to anterior cruciate ligament (ACL) fibroblasts, and it will hinder cell mobility and ACL regeneration, and even induce osteoarthritis. The mechano growth factor (MGF) could be responsible for mechanical stress and weakening its negative effects on cell physiological behaviors. In this study, effects of MGF on cell mobility and relevant molecules expression in injured ACL fibroblasts were detected. After an injurious mechanical stretch, the analysis carried out, at 0 and 24 h, respectively, showed that the cell area, roundness, migration, and adhesion of ACL fibroblasts were reduced. MGF (10, 100 ng/mL) treatment could improve cell area, roundness and promote cell migration and adhesion capacity compared with the injured group without MGF. Further study indicated that cell mobility-relevant molecules (PAK1/2, Cdc42, Rac1, RhoA, and ROCK1) expression in ACL fibroblasts was down-regulated at 0 or 24 h after injurious stretch (except Rac1 and RhoA at 0 h). Similarly, MGF improved cell mobility-relevant molecule expression, especially the ROCK1 expression level in ACL fibroblasts at 0 or 24 h after injurious stretch. Protein expression of ROCK1 in injured ACL fibroblasts was also reduced and could be recovered by MGF treatment. In a rabbit partial ACL transection (ACLT) model, ACL exhibited poor regenerative capacity in collagen and extracellular matrix (ECM) synthesis after partial ACLT for 2 or 4 weeks, and MGF remarkably accelerated ACL regeneration and restored its mechanical loading capacity after partial ACLT for four weeks. Our findings suggest that MGF weakens the effects of pathological stress on cell mobility of ACL fibroblasts and accelerates ACL repair, and might be applied as a future treatment approach to ACL rupture in the clinic.

Mechanically superior matrices promote osteointegration and regeneration of anterior cruciate ligament tissue in rabbits

The gold standard treatment for anterior cruciate ligament (ACL) reconstruction is the use of tendon autografts and allografts. Limiting factors for this treatment include donor site morbidity, potential disease transmission, and variable graft quality. To address these limitations, we previously developed an off-the-shelf alternative, a poly(l-lactic) acid (PLLA) bioengineered ACL matrix, and demonstrated its feasibility to regenerate ACL tissue. This study aims to 1) accelerate the rate of regeneration using the bioengineered ACL matrix by supplementation with bone marrow aspirate concentrate (BMAC) and growth factors (BMP-2, FGF-2, and FGF-8) and 2) increase matrix strength retention. Histological evaluation showed robust tissue regeneration in all groups. The presence of cuboidal cells reminiscent of ACL fibroblasts and chondrocytes surrounded by an extracellular matrix rich in anionic macromolecules was up-regulated in the BMAC group. This was not observed in previous studies and is indicative of enhanced regeneration. Additionally, intraarticular treatment with FGF-2 and FGF-8 was found to suppress joint inflammation. To increase matrix strength retention, we incorporated nondegradable fibers, polyethylene terephthalate (PET), into the PLLA bioengineered ACL matrix to fabricate a "tiger graft." The tiger graft demonstrated the greatest peak loads among the experimental groups and the highest to date in a rabbit model. Moreover, the tiger graft showed superior osteointegration, making it an ideal bioengineered ACL matrix. The results of this study illustrate the beneficial effect bioactive factors and PET incorporation have on ACL regeneration and signal a promising step toward the clinical translation of a functional bioengineered ACL matrix.

Knitted Silk-Collagen Scaffold Incorporated with Ligament Stem/Progenitor Cells Sheet for Anterior Cruciate Ligament Reconstruction and Osteoarthritis Prevention.

Current surgical management of anterior cruciate ligament (ACL) rupture still remains an intractable challenge in ACL regeneration due to the weak self-healing capability of ACL. Inadequate cell numbers and vascularization within the articular cavity contribute mainly to the poor prognosis. This time, we fabricated a new tissue engineering scaffold by adding ligament stem/progenitor cell (LSPC) sheets to our previous knitted silk-collagen sponge scaffold, which overcame these limitations by providing sufficient numbers of seed cells and a natural extracellular matrix to facilitate regeneration. LSPCs display excellent proliferation and multilineage differentiation capacity. Upon ectopic implantation, the knitted silk-collagen sponge scaffold incorporated with an LSPC sheet exhibited less immune cells but more fibroblast-like cells, deposited ECM and neovascularization, and better tissue ingrowth. In a rabbit model, we excised the ACL and performed a reconstructive surgery with our scaffold. Increased expression of ligament-specific genes and better collagen fibril formation could be observed after orthotopic transplantation. After 6 months, the LSPC sheet group showed better results on ligament regeneration and ligament-bone healing. Furthermore, no obvious cartilage and meniscus degeneration were observed at 6 months postoperation. In conclusion, these results indicated that the new tissue engineering scaffold can promote ACL regeneration and slow down the progression of osteoarthritis, thus suggesting its high clinical potential as an ideal graft in ACL reconstruction.

Osteointegration of a Novel Silk Fiber–Based ACL Scaffold by Formation of a Ligament-Bone Interface

Background: Given the unsatisfactory results and reported drawbacks of anterior cruciate ligament (ACL) reconstruction, such as donor site morbidity and the limited choice of grafts in revision surgery, new regenerative approaches based on tissue-engineering strategies are currently under investigation. Purposes: To determine (1) if a novel silk fiber–based ACL scaffold is able to initiate osteointegration in the femoral and tibial bone tunnels under in vivo conditions and (2) if the osteointegration process will be improved by intraoperatively seeding the scaffolds with the autologous stromal vascular fraction, an adipose-derived, stem cell–rich isolate from knee fat pads. Study Design: Controlled laboratory study. Methods: A total of 33 sheep underwent ACL resection and were then randomly assigned to 2 experimental groups: ACL reconstruction with a scaffold alone and ACL reconstruction with a cell-seeded scaffold. Half of the sheep in each group were randomly chosen and euthanized 6 months after surgery and the other half at 12 months. To analyze the integration of the silk-based scaffold in the femoral and tibial bone tunnels, hard tissue histology and micro–computed tomography measurements were performed. Results: Hard tissue histological workup showed that in all treatment groups, with or without the application of the autologous stromal vascular fraction, an interzone of collagen fibers had formed between bone and silk-based graft. This collagen-fiber continuity partly consisted of Sharpey fibers, comparable with tendon-bone healing known for autografts and allografts. Insertion sites were more broad based at 6 months and more concentrated on the slightly protruding, bony knoblike structures at 12 months. Histologically, no differences between the treatment groups were detectable. Analysis of micro–computed tomography measurements revealed a significantly higher tissue density for the cell-seeded scaffold group as compared with the scaffold-alone group in the tibial but not femoral bone tunnel after 12 months of implantation. Conclusion: The novel silk fiber–based scaffold for ACL regeneration demonstrated integration into the bone tunnels via the formation of a fibrous interzone similar to allografts and autografts. Histologically, additional cell seeding did not enhance osteointegration. No significant differences between 6 and 12 months could be detected. After 12 months, there was still a considerable amount of silk present, and a longer observation period is necessary to see if a true ligament-bone enthesis will be formed. Clinical Relevance: ACL regeneration with a silk fiber–based scaffold with and without additional cell seeding may provide an alternative treatment option to current techniques of surgical reconstruction.

MGF E peptide improves anterior cruciate ligament repair by inhibiting hypoxia-induced cell apoptosis and accelerating angiogenesis.

Severe hypoxic microenvironment endangers cell survival of anterior cruciate ligament (ACL) fibroblasts and is harmful to ACL repair and regeneration. In the current study, we explored the effects of mechanogrowth factor (MGF) E peptide on the hypoxia-induced apoptosis of ACL fibroblasts and relevant mechanisms. It demonstrated that severe hypoxia promoted hypoxia-inducible factor-1α (HIF-1α) expression and caused cell apoptosis of ACL fibroblasts through increasing caspase 3/7/9 messenger RNA (mRNA), cleaved caspase 3 and proapoptotic proteins expression levels but decreasing antiapoptotic proteins expression levels. Fortunately, MGF E peptide effectively protected ACL fibroblasts against hypoxia-induced apoptosis through regulating caspase 3/7/9 mRNA, cleaved caspase 3 and apoptosis-relevant proteins expression levels. Simultaneously, mitochondrial, @@@MEK-ERK1/2 (extracellular-signal-regulated kinase 1/2), and phosphoinositide-3-kinase-protein kinase B (PI3K-Akt) pathways were involved in MGF E peptide regulating hypoxia-induced apoptosis of ACL fibroblasts. In rabbit ACL rupture model, MGF E peptide also decreased HIF-1α expression levels, cell apoptosis, and facilitated cell proliferation. In addition, MGF could accelerate angiogenesis after ACL injury probably owing to its recruitment of proangiogenesis cells by stromal cell-derived factor 1α/CXCR4 axis and stimulation of vascular endothelial growth factor α expression level. In conclusion, our findings suggested that MGF E peptide could be utilized for ACL repair and regeneration and supplied experimental support for its application in clinical ACL treatment as a potential strategy.

Preclinical Studies on Biomaterial Scaffold use in Knee Ligament Regeneration: A Systematic Review.

Knee joint trauma may result in damage of the intra-articular ligaments, with rupture of the anterior cruciate ligament (ACL) a common and troublesome injury due to poor capabilities for spontaneous regeneration. Autograft and allograft surgical reconstructions are the mainstay of treatment, but have associated risks of failure, therefore tissue-engineering techniques aiming to regenerate the native ACL are being researched as a potential alternative treatment. This article aims to review the current evidence produced by ex vivo and in vivo studies investigating biomaterial scaffolding and mesenchymal stem cell (MSC) techniques in orthopaedic tissue engineering of ACL injuries. Databases searched were Ovid MEDLINE, Cochrane Library, Embase, Elsevier Scopus, Web of Science and NCBI PubMed, with search terms 'ligament', 'scaffold', 'mesenchymal stem cell' and 'tissue engineering'. 1132 articles were identified, with 19 articles suitable for review inclusion. Of the eligible studies, 10 used biologic scaffold material, 6 used synthetic constructs, and hybrid scaffolds were employed in the remaining 3 studies. A large amount of preclinical evidence for viability of MSC seeded biomaterial scaffolds in ACL regeneration exists. Studies show that with stimulation, MSCs adhere and proliferate well on various scaffold materials ranging from silk to engineered polymers. Hybrid scaffolds are particularly promising, and with further research, the best features from strong natural substances such as silk, and biologically inert synthetic materials could be combined. Currently, there are few plans to begin human clinical trials, but preclinical studies are moving into larger animal models.

Exogenous stromal derived factor-1 releasing silk scaffold combined with intra-articular injection of progenitor cells promotes bone-ligament-bone regeneration

Anterior cruciate ligament (ACL) is one of the most difficult tissues to heal once injured. Ligament regeneration and tendon-bone junction healing are two major goals of ACL reconstruction. This study aimed to investigate the synergistic therapeutic effects of Stromal cell-derived factor 1 (SDF-1)-releasing collagen-silk (CSF) scaffold combined with intra-articular injection of ligament-derived stem/progenitor cells (LSPCs) for ACL regeneration and the amelioration in the long-term complication of osteoarthritis (OA). The stem cell recruitment ability of CSF scaffold and the multipotency, particularly the tendon forming ability of LSPCs from rabbits were characterized in vitro, while the synergistic effect of the CSF scaffold and LSPCs for ACL regeneration and OA amelioration were investigated in vivo at 1, 3, and 6 months with a rabbit ACL reconstruction model. The CSF scaffold was used as a substitute for the ACL, and LSPCs were injected into the joint cavity after 7 days of the ACL reconstruction. CSF scaffold displayed a controlled release pattern for the encapsulated protein for up to 7 days with an increased stiffness in the mechanical property. LSPCs, which exhibited highly I Collagen and CXCR4 expression, were attracted by SDF-1 and successfully relocated into the CSF scaffold at 1 month in vivo. At 3 and 6 months post-treatment, the CSF scaffold combined with LSPCs (CSFL group) enhanced the regeneration of ACL tissue, and promoted bone tunnel healing. Furthermore, the OA progression was impeded efficiently. Our findings here provided a new strategy that using stem cell recruiting CSF scaffold with tissue-specific stem cells, could be a promising solution for ACL regeneration. Statement of SignificanceIn this study, we developed a silk scaffold with increased stiffness and SDF-1 controlled release capacity for ligament repair. This advanced scaffold transplantation combined with intra-articular injection of LSPCs (which was isolated from rabbit ligament for the first time in this study) promoted the regeneration of both the tendinous and bone tunnel portion of ACL. This therapeutic strategy also ameliorated cartilage degeneration and reduced the severity of arthrofibrosis. Hence, combining LSPCs injection with SDF-1-releasing silk scaffold is demonstrated as a therapeutic strategy for ACL regeneration and OA treatment in the clinic. [Display omitted]

Anterior cruciate ligament-derived mesenchymal stromal cells have a propensity to differentiate into the ligament lineage.

IntroductionThe anterior cruciate ligament (ACL) consists of various components, such as collagen, elastin fibres, and fibroblasts. Because ACL has a poor regenerative ability, ACL reconstruction need require the use of autologous tendons. In recent years, tissue-resident stem cells have been studied to promote ACL regeneration as an alternatively method. However, the existence of stem cells in ligaments has not been clearly defined. Here, we prospectively isolated stem cells from ACLs and characterized their properties.MethodsACLs from 11 donors and bone marrows (BM) from 8 donors were obtained with total knee arthroplasty. We used flow cytometry to screen the cell surface markers on ACL cells. Frozen sections were prepared from patient ACL tissues and stained with specific antibodies. Cultured ACL-derived and BM-derived cells at passage 3 were differentiated into adipocytes, osteoblasts and tendon/ligament cells.ResultsACL-derived mesenchymal stem/stromal cells (ACL-MSCs) expressed high levels of CD73 and CD90. Immunohistochemical analyses revealed that ACL-MSCs were located on the inner surface of ACL sinusoids. Furthermore, the expression of cell surface antigens was clearly different between ACL-MSCs and bone marrow (BM)-derived MSCs (BM-MSCs) at the time of isolation, but the two cell populations became indistinguishable after long-term culture. Interestingly, ACL-MSCs are markedly different from BM-MSCs in their differentiation ability and have a high propensity to differentiate into ligament-committed cells.ConclusionsOur findings suggest that ACL-MSCs express CD90 and CD73 markers, and their differentiation capacity is maintained even through culture. The cell population having tissue-specific properties is an important research target for investigating the ligament therapies.

Design and characterization of three-dimensional twist-braid scaffolds for anterior cruciate ligament regeneration

The anterior cruciate ligament (ACL) is the most commonly injured ligament in the knee, with more than 350,000 ACL injuries reported annually in the US. Current treatments include the use of autografts and allografts, which have a number of disadvantages. Previous attempts to use synthetic materials in ligament replacement have been unsuccessful due to their inability to replicate the long-term mechanical properties of the native ligament. The focus of this study was to develop twist-braid poly(L-lactic acid) (PLLA) scaffolds for ACL regeneration. Poly(ethylene glycol) diacrylate (PEGDA) was incorporated into the twist-braid scaffolds to evaluate its impact on their mechanical behavior. The twist-braid scaffolds were also compared with braided scaffolds. Scaffold mechanical properties were evaluated based on stress-relaxation, tensile and fatigue properties of the braided-only, twist-braid, and the twist-braid scaffolds with PEGDA. All the scaffolds exhibited properties comparable to the native human ACL with the twist-braid scaffolds displaying resistance to fatigue. Scaffolds were seeded with rat patellar tendon fibroblasts. Cell viability and the amount of protein released were studied over a course of 8 weeks. The scaffolds were stained with Picrosirius red after 8 weeks to show the deposition of extracellular matrix by the cells. The results from this study showed that the twist-braid scaffolds have properties most suitable for ligament regeneration.

Ectopic tissue engineered ligament with silk collagen scaffold for ACL regeneration: A preliminary study.

Early inflammatory cell infiltration, tissue and vessels ingrowth were significantly higher in the extra-articular implanted scaffolds than theses in the joint cavity. By mimicking the early stages of wound repair, which provided extra-articular inflammatory stimulation to facilitate the early ingrowth of blood vessels and cells, a vascularized ectopic tissue engineered ligament (ETEL) with silk collagen scaffold was constructed by subcutaneous implantation for 2weeks. The fully vascularized TE ligament was then transferred to rebuild ACL without blood perfusion interruption, and was demonstrated to exhibit improved ACL regeneration, bone tunnel healing and mechanical properties.

MGF E peptide pretreatment improves collagen synthesis and cell proliferation of injured human ACL fibroblasts via MEK-ERK1/2 signaling pathway

Injured anterior cruciate ligament (ACL) is hard to heal due to the poor proliferative potential of ACL fibroblasts. To verify whether mechano-growth factor (MGF) E peptide can restore the cell proliferation of injured ACL fibroblasts, ACL fibroblasts pretreated with MGF E peptide were subjected to injurious stretch and the outcomes were evaluated at 0 and 24 h. After injured, the type III collagen synthesis was increased at 0 h while inhibited at 24 h. The matrix metalloproteinase-2 (MMP-2) activity/expression was up-regulated, but the cell proliferation was inhibited. Fortunately, exogenous MGF E peptide decreased the type I/III collagen synthesis at 0 h but improved the type III collagen synthesis at 24 h. It decreased the MMP-2 activity/expression of injured ACL fibroblasts. Besides, MGF E peptide accelerated the cell proliferation via MEK-ERK1/2 signaling pathway. Our results implied that MGF E peptide pretreatment could provide a new efficient approach for ACL regeneration.

Microfiber-reinforced nanofibrous scaffolds with structural and material gradients to mimic ligament-to-bone interface.

Most of the existing tissue-engineered scaffolds for the regeneration of anterior cruciate ligament (ACL) are unable to recapitulate the structural/compositional/mechanical gradients at native ACL-bone interface. Herein, we present a unique electrospinning strategy to fabricate microfiber-reinforced nanofibrous scaffolds with structure and composition gradients. The orientations of the electrospun nanofibers gradually change from random to highly aligned, and the contents of nano-hydroxyapatite (nHA) and bone morphogenesis protein (BMP-2) encapsulated into the nanofibers exhibit gradient variation. The gradient nanofibrous scaffolds were found to regulate cellular morphology and zonal bone-specific differentiation in vitro. The incorporation of microfibers significantly improved the mechanical property of the gradient nanofibrous scaffolds, which was similar to native ACL-bone fixation after implantation in vivo. Histological observations revealed that the gradient scaffolds facilitated the formation of fibrocartilage transitional zone at graft and bone attachments that are similar to native ligament-to-bone interface. We envision that the presented method might provide biomimetic physical/chemical/mechanical microenvironments for enhanced multitissue regeneration particularly at the soft-hard tissue interface.

A Novel Silk Fiber–Based Scaffold for Regeneration of the Anterior Cruciate Ligament

Background: Because of ongoing problems with anterior cruciate ligament (ACL) reconstruction, new approaches in the treatment of ACL injuries, particularly strategies based on tissue engineering, have gained increasing research interest. To allow for ACL regeneration, a structured scaffold that provides a mechanical basis, has cells from different sources, and comprises mechanical as well as biological factors is needed. Biological materials, biodegradable polymers, and composite materials are being used and tested as scaffolds. The optimal scaffold for ACL regeneration should be biocompatible and biodegradable to allow tissue ingrowth but also needs to have the right mechanical properties to provide immediate mechanical stability. Hypotheses: The study hypotheses were that (1) a novel degradable silk fiber–based scaffold with mechanical properties similar to the native ACL will be able to initiate ligament regeneration after ACL resection and reconstruction under in vivo conditions and (2) additional cell seeding of the scaffold with autologous stromal vascular fraction–containing adipose-derived stem cells will increase regenerative activity. Study Design: Controlled laboratory study. Methods: A total of 33 mountain sheep underwent ACL resection and randomization to 2 experimental groups: (1) ACL reconstruction with a scaffold alone and (2) ACL reconstruction with a cell-seeded scaffold. Histological evaluation of the intra-articular portion of the reconstructed/regenerated ligament was performed after 6 and 12 months. Results: After 6 months, connective tissue surrounded the silk scaffold with ingrowth in some areas. The cell-seeded scaffolds had a significant lower silk content compared with the unseeded scaffolds and demonstrated a higher content of newly formed tissue. After 12 months, the density of the silk fibers decreased significantly, and the ingrowth of newly formed tissue increased in both groups. No differences between the 2 groups regarding silk fiber degradation and regenerated tissue were detected at 12 months. Conclusion: The novel silk fiber–based scaffold was able to stimulate ACL regeneration under in vivo conditions. Additional cell seeding led to increased tissue regeneration and decreased silk fiber content at 6 months, whereas these differences were not present at 12 months. Clinical Relevance: ACL regeneration using a silk fiber–based scaffold with and without additional cell seeding may provide a new treatment option after joint injuries.

Evaluation of polycaprolactone scaffold with basic fibroblast growth factor and fibroblasts in an athymic rat model for anterior cruciate ligament reconstruction.

Anterior cruciate ligament (ACL) rupture is a common ligamentous injury often necessitating surgery. Current surgical treatment options include ligament reconstruction with autograft or allograft, which have their inherent limitations. Thus, there is interest in a tissue-engineered substitute for use in ACL regeneration. However, there have been relatively few in vivo studies to date. In this study, an athymic rat model of ACL reconstruction was used to evaluate electrospun polycaprolactone (PCL) grafts, with and without the addition of basic fibroblast growth factor (bFGF) and human foreskin fibroblasts. We examined the regenerative potential of tissue-engineered ACL grafts using histology, immunohistochemistry, and mechanical testing up to 16 weeks postoperatively. Histology showed infiltration of the grafts with cells, and immunohistochemistry demonstrated aligned collagen deposition with minimal inflammatory reaction. Mechanical testing of the grafts demonstrated significantly higher mechanical properties than immediately postimplantation. Acellular grafts loaded with bFGF achieved 58.8% of the stiffness and 40.7% of the peak load of healthy native ACL. Grafts without bFGF achieved 31.3% of the stiffness and 28.2% of the peak load of healthy native ACL. In this in vivo rodent model study for ACL reconstruction, the histological and mechanical evaluation demonstrated excellent healing and regenerative potential of our electrospun PCL ligament graft.

REGENERATION OF ANTERIOR CRUCIATE LIGAMENT WITH SILK-BASED SCAFFOLD IN PORCINE MODEL

Silk was widely investigated as a promising scaffold material in ligament tissue engineering. Although a variety of silk scaffolds were developed for the regeneration of anterior cruciate ligament (ACL) in vitro and in vivo, more investigations should be performed in large animals to translate these findings into clinical applications. The aim of this study is to evaluate the feasibility of using silk-based ACL scaffolds to regenerate damaged ACLs in porcine model. The microstructural organization, tissue regeneration as well as ligament-bone interface of silk implants were evaluated with scanning electron microscopy, micro-computerized tomography, histological and immunohistochemical staining at three and six months postoperatively. The results demonstrated that silk fibers in the ACL scaffolds organized in parallel similar with collagen fibers in native ligaments, which facilitated and guided the penetration of newly regenerated tissue into the pores among silk fibers. Collagen production especially collagen I in silk implants significantly increased from three to six months, and was gradually close to the level of native ligaments. At implant-bone interface, indirect ligament-bone insertion was observed at three months and substantial Sharpey's fibers formed at six months. The results indicated that the silk-based ACL scaffold provides a promising tissue engineering approach for ACL regeneration.