Introduction: Osteochondral lesions of the talus (OCL III–IV°) need both extensive debridement for revitalisation and osteochondral reconstruction of the joint surface. This can be achieved by autologous cancellous bone-grafting and combination with a cell-free bioresorbable collagen-I/III scaffold. Our first results with this technique are presented. Methods: 25 patients (13 female, 12 male, mean age 30.9 years) with 26 osteochondral lesions of the talus (OCL III–IV°, 15 right, 11 left, 24 medial, 2 lateral, 1 bilateral case) were treated by minimal-invasive debridement, autologous cancellous bone-grafting and application of a porcine collagen-I/III scaffold (ChondroGide. ®. ) and evaluated prospectively by clinical scoring and MRI. The average follow-up was 23.2 (6–36) months. The mean defect size was 2.0 cm. 2. , the mean depth 0.7 cm. 14 defects have had at least one (1–3) operation on the defect before. By the use of a distractor a malleolar osteotomy could be avoided in all cases. Results: The AOFAS-score increased from 67.4 ± 12.2 to 89.5 ± 7.4 points (p< 0.01, t-test). On a visual 10-point scale pain decreased significantly from 6.2 to 1.7 while subjective ankle function improved from a mean of 4.4 ± 1.9 to 7.2 ±1.5. The results were rated excellent in 10/26 cases (38.4%), good in 14/26 (53.8) and fair in 2/26 (7.8%) cases. MRI follow-ups showed a complete or nearly complete defect filling. In two ankles a second-look arthroscopy unveiled the defects filled completely by a regenerative tissue with a smooth surface and good bonding. Full-core biopsies showed a mixed, mostly fibrocartilagenous tissue. Conclusion: By combination of cancellous bone-grafting with a cell-free collagen-I/III scaffold typical osteochondral lesions of the talus can be adressed effectively in a minimal-invasive one-step procedure. By utilizing mesenchymal stem cells (MSC) for an autogenous reparation process the use of expensive cultured chondrocytes is not necessary. The results concerning clinical functional improvement, pain reduction and patients’ satisfaction as well as defect filling in MRI are promising

Treatment of segmental bone loss remains a major challenge in orthopaedic surgery. This study evaluated the healing potential of a series of highly porous tissue engineering scaffolds with the current clinical gold standard. We compare healing of collagen-glycosaminoglycan (CG) and collagen micro-hydroxyapatite (CHA) scaffolds, with and without recombinant bone morphogenetic protein-2 (BMP2), with autogenous bone graft (ABG) in the healing of a 15mm rabbit radius defect, which were filled with either CG scaffold, CHA scaffold, CG-BMP2, CHA-BMP2 or ABG. Serial radiographs and micro-computed tomography (µCT) at six week radiographs demonstrated complete defect bridging with callus using CHA and CG-BMP2 while the CHA-BMP2 was already in an advanced state of healing with cortical remodeling. By sixteen weeks CHA, CG-BMP2 and ABG all had advanced healing with cortical remodeling while CHA-BMP2 had complete anatomic healing. Quantitative histomorphometry values demonstrated similarly high healing levels of healing in CHA, CG-BMP2 and ABG with highest overall values in the CHA-BMP2 group. Thus, treatment of a critical sized, weight bearing, rabbit radius defect with a CHA scaffold can result in full cortical bridging with medullary cavity development. In addition, a CHA-BMP2 combination can result in fully mature, anatomic healing. The use of an off-the-shelf CHA scaffold for direct surgical placement into a defect site may be an effective bone graft substitute in the treatment of skeletal defects. The ease of manufacture, storage and peri-operative preparation may offer an alternative to traditional strategies, as well as to more recent BMP2 devices. This study provides clear evidence that CHA scaffolds can perform as well as autogenous bone grafts and supports their use as a viable alternative. Where the use of BMP2 may be desirable, these materials provide an ideal delivery mechanism and using a very low (near physiological) dose, healing superior to autogenous graft may be achieved

Cartilage injuries often represent irreversible tissue damage because cartilage has only a low ability to regenerate. Thus, cartilage loss results in permanent damage, which can become the starting point for osteoarthritis. In the past, bioactive glass scaffolds have been developed for bone replacement and some of these variants have also been colonized with chondrocytes. However, the hydroxylapaptite phase that is usually formed in bioglass scaffolds is not very suitable for cartilage formation (chondrogenesis). This interdisciplinary project was undertaken to develop a novel slowly degrading bioactive glass scaffold tailored for cartilage repair by resembling the native extracellular cartilage matrix (ECM) in structure and surface properties. When colonized with articular chondrocytes, the composition and topology of the scaffolds should support cell adherence, proliferation and ECM synthesis as a prerequisite for chondrogenesis in the scaffold. To study cell growth in the scaffold, the scaffolds were colonized with human mesenchymal stromal cells (hMSCs) and primary porcine articular chondrocytes (pACs) (27,777.8 cells per mm. 3. ) for 7 – 35 d in a rotatory device. Cell survival in the scaffold was determined by vitality assay. Scanning electron microscopy (SEM) visualized cell ultramorphology and direct interaction of hMSCs and pACs with the bioglass surface. Cell proliferation was detected by CyQuant assay. Subsequently, the production of sulphated glycosaminoglycans (sGAGs) typical for chondrogenic differentiation was depicted by Alcian blue staining and quantified by dimethylmethylene blue assay assay. Quantitative real-time polymerase chain reaction (QPCR) revealed gene expression of cartilage-specific aggrecan, Sox9, collagen type II and dedifferentiation-associated collagen type I. To demonstrate the ECM-protein synthesis of the cells, the production of collagen type II and type I was determined by immunolabelling. The bioactive glass scaffold remained stable over the whole observation time and allowed the survival of hMSCs and pACs for 35 days in culture. The SEM analyses revealed an intimate cell-biomaterial interaction for both cell types showing cell spreading, formation of numerous filopodia and ECM deposition. Both cell types revealed initial proliferation, decreasing after 14 days and becoming elevated again after 21 days. hMSCs formed cell clusters, whereas pACs showed an even distribution. Both cell types filled more and more the pores of the scaffold. The relative gene expression of cartilage-specific markers could be proven for hMSCs and pACs. Cell associated sGAGs deposition could be demonstrated by Alcian blue staining and sGAGs were elevated in the beginning and end of the culturing period. While the production of collagen type II could be observed with both cell types, the synthesis of aggrecan could not be detected in scaffolds seeded with hMSCs. hMSCs and pACs adhered, spread and survived on the novel bioactive glass scaffolds and exhibited a chondrocytic phenotype

Introduction. The objective of the work is construction of a multi-bioactive scaffold based on that allows a space/time control over the regeneration of damaged bones by Medication-Related Osteonecrosis of the Jaw using a minimal invasive approach based on the injection of the fast-degrading pro neuro and angiogenic ELR (Elastin-Like Recombinamers) based hydrogels. Method. Chemical crosslinking facilitated the creation of multi-bioactive scaffolds using ELRs with reactive groups. Cell-loaded multi-bioactive scaffolds, prepared and incubated, underwent evaluation for adhesion, proliferation, angiogenic, and neurogenic potential. In vitro assessments utilized immunofluorescence staining and ELISA assays, while live-recorded monitoring and live-dead analysis ensured cytocompatibility. In rat and rabbit models, preformed scaffolds were subcutaneously implanted, and the regenerative process was evaluated over time. Rabbit models with MRONJ underwent traditional or percutaneous implantation, with histological evaluation following established bone histological techniques. Result. A 3D scaffold using ELR that combines various peptides with different degradation rates to guide both angiogenesis and neurogenesis has been developed. Notably, scaffolds with different degradation rates promoted distinct patterns of vascularization and innervation, facilitating integration with host tissue. This work demonstrates the potential for tailored tissue engineering, where the scaffold's bioactivities and degradation rates can control angiogenesis and neurogenesis. In an animal model of medication-related osteonecrosis of the jaw (MRONJ), the scaffold showed promising results in promoting bone regeneration in a necrotic environment, as confirmed by histological and imaging analyses. This study opens avenues for novel tissue-engineering strategies where precise control over vascularization and nerve growth is crucial. Conclusion. A groundbreaking dual approach, simultaneously targeting angiogenesis and innervation, addresses the necrotic bone in MRONJ syndrome. Vascularization and nerve formation play pivotal roles in driving reparative elements for bone regeneration. The scaffold achieves effective time/space control over necrotic bone regeneration. The authors are grateful for funding from the Spanish Government (PID2020-118669RA-I00)

Introduction and Objective. Regeneration of cartilage injuries is greatly limited. Therefore, cartilage injuries are often the starting point for later osteoarthritis. In the past, various bioactive glass (BG) scaffolds have been developed to promote bone healing. Due to the fact that they induce the deposition of hydroxyapatite (HA) -the main component of bone matrix, these BG types are not suitable for chondrogenesis. Hence, a novel BG (Car12N) lacking HA formation, was established. Since BG are generally brittle the combination with polymers is helpful to achieve suitable biomechanic stability. The aim of this interdisciplinary project was to investigate the effects of biodegradable polymer Poly(D,L-lactide-co-glycolide) (PLLA) infiltration into a Car12N scaffold for cartilage tissue engineering. Materials and Methods. BG scaffolds were infiltrated with PLLA using phase separation within a solvent. Pure BG Car12N scaffolds served as control. To assess whether the polymer was homogeneously distributed the polymer to glass ratio and pore contents in the upper, middle and lower third of the scaffolds were examined by light microscopy. For a more precise characterization of the scaffold topology, the glass strut length, the glass strut diameter and the pore circumference were also measured. Leaching tests in 0.1M HCl solution over 8 days were used to allow a gel layer formation on the scaffolds surface. Non-leached and leached scaffolds were subjected to strength testing. Cytotoxicity of the scaffolds with and without polymer was tested according to standards. Scaffolds were colonized with 27.777.8 per cm. 3. primary porcine articular chondrocytes (pACs) or primary human mesenchymal stromal cells (hMSCs), respectively. After cultivation for up to 35 days, the vitality, quantitative DNA and sulfated glycosaminoglycan (sGAG) contents per scaffold were determined. Results. The polymer distribution was not homogeneous in the scaffolds. There were significant differences in glass strut length and pore size. Leaching increased the biomechanical strength. All scaffolds were not cytotoxic. pACs and hMSCs were able to adhere to the scaffold with and without polymer and remained viable during the whole culturing period of 35 d. The DNA content was higher in the pAC colonized scaffolds with polymer than without polymer. The sGAG content was higher in hMSCs seeded scaffolds with polymer than in pACs seeded ones with polymer. Conclusions. Polymer infiltration leads to an increase in mechanical stability of Car12N scaffolds and chondrogenic cells are able to colonize these composites suggesting them as a promising

Even minor lesions in articular cartilage (AC) can cause underlying bone damage creating an osteochondral (OC) defect. OC defects can cause pain, impaired mobility and can develop to osteoarthritis (OA). OA is a disease that affects nearly 10% of the population worldwide. [1]. , and represents a significant economic burden to patients and society. [2]. While significant progress has been made in this field, realising an efficacious therapeutic option for unresolved OA remains elusive and is considered one of the greatest challenges in the field of orthopaedic regenerative medicine. [3]. Therefore, there is a societal need to develop new strategies for AC regeneration. In recent years there has been increased interest in the use of tissue-specific aligned porous freeze-dried extracellular matrix (ECM) scaffolds as an off-the-shelf approach for AC repair, as they allow for cell infiltration, provide biological cues to direct target-tissue repair and permit aligned tissue deposition, desired in AC repair. [4]. However, most ECM-scaffolds lack the appropriate mechanical properties to withstand the loads passing through the joint. [5]. One solution to this problem is to reinforce the ECM with a stiffer framework made of synthetic materials, such as polylactic acid (PLA). [6]. Such framework can be 3D printed to produce anatomically accurate implants. [7]. , attractive in personalized medicine. However, typical 3D prints are static, their design is not optimized for soft-hard interfaces (OC interface), and they may not adapt to the cyclic loading passing through our joints, thus risking implant failure. To tackle this limitation, more compliant or dynamic designs can be printed, such as coil-shaped structures. [8]. Thus, in this study we use finite element modelling to create different designs that mimic the mechanical properties of AC and prototype them in PLA, using polyvinyl alcohol as support. The optimal design will be combined with an ECM scaffold containing a tailored microarchitecture mimicking aspects of native AC. Acknowledgments: This project has received funding from the European Union's Horizon Europe research and innovation MSCA PF programme under grant agreement No. 101110000

Abstract. Objectives. Assess and characterise the suitability of a novel silk reinforced biphasic 3D printed scaffold for osteochondral tissue regeneration. Methods. Biphasic hybrid scaffolds consisted of 3D printed poly(ethylene glycol)-terephthalate-poly(butylene terephthalate)(PEGT/PBT) scaffold frame work (pore size 0.75mm), which has been infilled with a cast and freeze dried porous silk scaffold (5×5×2mm. 3. ), in addition to a seamless silk top layer (1mm). Silk scaffolds alone were used as controls. Both the biphasic and control scaffolds were characterised via uniaxial compression testing (strain rate 0.1mm/min), and the potential biocompatibility of the scaffolds was tested via in vitro culture of seeded bone marrow stromal cells post fabrication. Results. Uniaxial compression testing showed that the biphasic scaffolds (N=4) initially demonstrated similar behaviour on a stress-strain curve to a silk scaffold alone control group (N=6), until a strain of 30% was reached. After 30% strain, load was transitioned from the silk only chondral layer to the 3D printed PEGT/PBT scaffold which resisted further compression and exhibited a significantly greater compressive modulus of 12.6±0.9MPa compared to 0.113±0.01MPa (p<0.001) in the silk scaffold control group. Following 24hours of seeding, no difference was noticed in cell adhesion behaviour under fluorescent microscopy between silk scaffolds and biphasic scaffolds (n=5). Discussion. The use of 3D printing within this novel scaffold provides a solid framework and increases its versatility. The reinforced silk not only provides the secondary Porous structure to the 3D printed scaffold for the bone phase, but also a superficial layer for the cartilage phase. This unique structure has the potential to fill a niche within osteochondral tissue regeneration, especially with the possibility for its use within personalised medicine. Conclusions. These results demonstrate that the novel silk reinforced biphasic 3D printed scaffold is biocompatible and has suitable mechanical properties for osteochondral tissue regeneration. Declaration of Interest. (b) declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported:I declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research project

The enthesis is a specialised zonal tissue interface between tendon and bone, essential for adequate force transmission and composed by four distinct zones, namely tendon, fibrocartilage, mineralized fibrocartilage and bone. Following injuries and surgical repair, the enthesis is often not reestablished and so far, traditionally used tissue substitutes have lacked to reproduce the complexity of the native tissue. In this work, we hypothesised that a collagen-based three-layer scaffold that mimic the composition of the enthesis, in combination with bioactive molecules, will enhance the functional regeneration of the enthesis. A three-layer sponge composed of a tendon-like layer (collagen I), a cartilage-like layer (collagen II) and a bone-like layer (collagen I and hydroxyapatite) was fabricated by an iterative layering freeze-drying technique. Scaffold porosity and structural continuity at the interfaces were assessed through SEM analysis. Bone-marrow derived stem cells (BMSCs) were seeded by syringe vacuum assisted technique on the scaffold. Scaffolds were cultured in basal media for 3 days before switching to differentiation media (chondrogenic, tenogenic and osteogenic). BMSCs metabolic activity, proliferation and viability were assessed by alamarBlue, PicoGreen and Live/Dead assays. At D21 the scaffolds were fixed, cryosectioned and Alizarin Red and Alcian Blue stainings were performed in order to evaluate BMSC differentiation towards osteogenic and chondrogenic lineage. The presence of collagen I and tenascin in the scaffolds was evaluated by immunofluorescence staining at D21 in order to assess tenogenic differentiation of BMSCs. Subsequently, the cartilage-like layer was functionalized with IGF-1, seeded with BMSCs and cultured in basal media up to D21. Structural continuity at the interfaces of the scaffolds was confirmed by SEM and scaffold porosity was assessed as >98%. The scaffolds supported cell proliferation and infiltration homogeneously throughout all the layers up to D21. Osteogenic differentiation of BMSC selectively in the bone-like layer was confirmed by Alizarin red staining in scaffolds cultured in basal and osteogenic media. Alcian blue staining revealed the presence of proteoglycans selectively in the cartilage-like layer in scaffolds cultured in chondrogenic media but not in basal media. Increased expression of the tenogenic markers collagen I and tenascin were observed in the tendon-like layer of scaffolds cultured in tenogenic but not in basal media for 21 days. The presence of IGF-1 increased osteogenic and chondrogenic differentiation of BMSCs, whereas no difference was observed for tenogenic differentiation. In conclusion, a 3-layer collagen sponge was successfully fabricated with distinct but integrated layers; the different collagen composition of the non-functionalized 3-layer sponge was able to regulate BMSC differentiation in a localized manner within the scaffold. The scaffold functionalization with IGF-1 accelerated chondrogenic and osteogenic BMSC differentiation. Overall, functionalization of the 3-layer scaffolds holds promising potential in enthesis regeneration

Critical size bone defects deriving from large bone loss are an unmet clinical challenge1. To account for disadvantages with clinical treatments, researchers focus on designing biological substitutes, which mimic endogenous healing through osteogenic differentiation promotion. Some studies have however suggested that this notion fails to consider the full complexity of native bone with respect to the interplay between osteoclast and osteoblasts, thus leading to the regeneration of less functional tissue2. The objective of this research is to assess the ability of our laboratory's previously developed 6-Bromoindirubin-3’-Oxime (BIO) incorporated guanosine diphosphate crosslinked chitosan scaffold in promoting multilineage differentiation of myoblastic C2C12 cells and monocytes into osteoblasts and osteoclasts1, 3, 4. BIO addition has been previously demonstrated to promote osteogenic differentiation in cell cultures5, but implementation of a co-culture model here is expected to encourage crosstalk thus further supporting differentiation, as well as the secretion of regulatory molecules and cytokines2. Biocompatibility testing of both cell types is performed using AlamarBlue for metabolic activity, and nucleic acid staining for distribution. Osteoblastic differentiation is assessed through quantification of ALP and osteopontin secretion, as well as osteocalcin and mineralization staining. Differentiation into osteoclasts is verified using SEM and TEM, qPCR, and TRAP staining. Cellular viability of C2C12 cells and monocytes was maintained when cultured separately in scaffolds with and without BIO for 21 days. Both scaffold variations showed a characteristic increase in ALP secretion from day 1 to 7, indicating early differentiation but BIO-incorporated sponges yielded higher values compared to controls. SEM and TEM imaging confirmed initial aggregation and fusion of monocytes on the scaffold's surface, but BIO addition appeared to result in smoother cell surfaces indicating a change in morphology. Late-stage differentiation assessment and co-culture work in the scaffold are ongoing, but initial results show promise in the material's ability to support multilineage differentiation. Acknowledgements: The authors would like to acknowledge the financial support of the Collaborative Health Research Program (CHRP) through CIHR and NSERC, as well as Canada Research Chair – Tier 1 in Regenerative Medicine and Nanomedicine, and the FRQ-S

Due to unsatisfactory results and reported drawbacks of anterior cruciate ligament (ACL) reconstruction new regenerative approaches based on tissue-engineering strategies are currently under investigation. It was the purpose of this study to determine if a novel silk fiber-based ACL scaffold is able to initiate osteointegration in the femoral and tibial bone tunnels under in vivo conditions. Furthermore we tested 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. In this controlled laboratory study, 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. The 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. 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

Silk fibroin (SF) has been used as a scaffold for cartilage tissue engineering. Different silkworms strain produced different protein. Also, molecular weight of SF depends on extraction method. We hypothesised that strain of silkworm and method of SF extraction would effect biological properties of SF scaffold. Therefore, cell viability and chondrogenic gene expression of human chondrogenic progenitor cells (HCPCs) treated with SF from 10 silkworm strains and two common SF extraction methods were investigate in this study. Twenty g of 10 strains silk cocoons were separately degummed in 0.02M Na2CO3 solution and dissolved in 100๐C for 30 minutes. Half of them were then dissolved in CaCl2/Ethanol/H2O [1:2:8 molar ratio] at 70±5๐C (method 1) and other half was dissolved in 46% w/v CaCl2 at 105±5๐C (method 2) for 4 hours. HCPCs were cultured in SF added cultured medial according to strain and extraction method. Cell viability at day 1, 3, and 7, were determined. Expression of collagen I, collagen II, and aggrecan at day 7 and 14, was studied. All experiment were done in triplicated samples. Generally, method 1 SF extraction showed higher cell viability in all strains. Cell viability from Nanglai Saraburi, Laung Saraburi and Nangtui strains were higher than those without SF in every time point while Wanasawan and J108 had higher viability at day 1 and decreased by time. Expression in collagen 1, collagen 2 and aggrecan in method 1 are higher at day 7 and day 14. Collagen 1 expression was highest in Nangnoi Srisaket, followed by Laung Saraburi and Nanglai Saraburi in day 7. Nangnoi Srisaket also had highest expression at day 14, followed by Nanglai Saraburi and Laung Saraburi respectively. Nangseaw had highest collagen 2 expression, follow by Laung Saraburi and Nangnoi Srisaket respectively. Higher aggrecan gene expression of Tubtimsiam, Wanasawan, UB 1 and Nangnoi Srisaket was observed at day 7 and increased expression of all strains at day 14. SF extraction using CaCl2/Ethanol/H2O offered better cell viability and chondrogenic expression. Nangseaw, Laung Saraburi and Nangnoi Srisaket strains expressed more chondrogenic phenotype

The need for an artificial scaffold in very large bone defects is clear, not only to limit the risk of graft harvesting, but also to improve clinical success. The use of custom osteoconductive scaffolds made from biodegradable polyester and ceramics can be a valuable patient friendly option, especially in case of a concomitant infection. Multiple types of scaffolds for the Masquelet procedure (MP) are available, however these frequently demonstrate central graft involution when defects exceed a certain size and the complication rates remains high. This paper describes three infected tibial defect nonunions with a segmental defect over ten centimeters long treated with a customized 3D printed polycaprolactone-tricalcium phosphate (PCL-TCP) cage in combination with biological adjuncts. Three male patients, between the age of 37 and 47, were treated for an infected tibial defect nonunion after sustaining Gustilo grade 3 open fractures. All had a segmental midshaft bone defect of more than ten centimeters (range 11–15cm). First stage MPs consisted of extensive debridement, external fixation and placement of anterior lateral thigh flaps (ALT). Positive cultures were obtained from all patients during this first stage, that were treated with specific systemic antibiotics during 12 weeks. The second stage MP was carried out at least two months after the first stage. CT scans were obtained after the first stage to manufacture defect-specific cages. In the final procedure a custom 3D printed PCL-TCP cage (Osteopore, Singapore) was placed in the defect in combination with biological adjuncts (BMAC, RIA derived autograft, iFactor and BioActive Glass). Bridging of the defect, assessed at six months by CT, was achieved in all cases. SPECT-scans 6 months post-operatively demonstrated active bone regeneration, also involving the central part of the scaffold. All three patients regained function and reported less pain with full weight-bearing. This case report shows that 3D printed PCL-TCP cages in combination with biological adjuncts is a novel addition to the surgical treatment of very large bone defects in (infected) posttraumatic nonunion of the tibia. This combination could overcome some of the current drawbacks in this challenging indication

For the treatment of irreparable meniscal injuries, we developed a novel multilayer meniscal scaffold, consisting of collagen, strontium and cellulose derived from Luffa Cylindirica; and we evaluated its effects on meniscal regeneration and arthritic changes in a rabbit partial meniscectomy model. The meniscus has a key role in shock absorbtion, load distribution, chondroprotection and stability of the knee joint. Meniscal injuries are one of the most common orthopedic injuries and may lead to degenerative cartilage changes and eventually osteoarthritis. Repair of the meniscal tissue is the treatment of choice for patients with a meniscus lesion, however, this is not always possible, especially for degenerative tears or injuries located on the inner avascular zone. To overcome the devastating outcomes of meniscectomy for such injuries, several materials have been developed and tried to replace the resected meniscal tissue. These scaffolds were designed primarily to relieve pain after meniscectomy, and later on were aimed to prevent osteoarthritis and cartilage damage that may develop in the future. In the quest for optimum scaffold material small intestine, tendons and other isolated tissues, collagen and polyurethane have been researched. Nevertheless, none of these materials have absolutely proven satisfying identical replacement of resected meniscal tissue. Therefore, we developed and investigated a novel multilayer meniscal scaffold, consisting of collagen, strontium and cellulose derived from Luffa Cylindirica (a cucumber shaped and sized plant, known as sponge gourd). The aim of the study was to evaluate the meniscal regeneration and arthritic changes after partial meniscectomy and application of novel multilayer meniscal scaffold in a rabbit model and to compare the results with clinically used polyurethane scaffold (Actifit, Orteq Ltd, London, UK). Sixteen male, mature, NewZealand rabbits weighing between 2600–3500 g were randomly divided into three groups. All groups underwent knee surgery via a medial parapatellar approach and a reproducible 1.5-mm cylindrical defect was created in the avascular zone of the anterior horn of the medial meniscus bilaterally. Defects were filled with the polyurethane scaffold in Group 1 and novel multilayer scaffold was applied to fill the defects in Group 2(n:6). Four rabbits in Group 3 did not receive any treatment and defects were left empty. Animals were sacrified after 8 weeks and bilateral knee joints were taken for macroscopic, biomechanical, and histological analysis. No signs of inflammation or infection were observed in all animals. Macroscopic evaluation of tibial plateaus after excision of menisci was performed with digital images of inked condylar surfaces. No significant degenerative changes were detected between groups. Digital photographs of excised menisci were also obtained and surface areas were measured by a computer software (Image J version 1.46, National Institute of Health, Bethesda, MD). There was a slightly larger meniscus area in the first two groups than the no treatment group, however, this was not found significant. Indentation testing of the tibial condyle and compression tests for the relevant meniscal areas with a diameter of 3mm was also performed in all groups. Histological analysis was made and all specimens were stained with safranin O and scored according to a scoring system. In this study, the initial evaluation of novel multilayer meniscal scaffold demonstrated promising biomechanical and histological results; besides no adverse events related to scaffold material was observed

In 2021 the bone grafting market was worth €2.72 billion globally. As allograft bone has a limited supply and risk of disease transmission, the demand for synthetic grafting substitutes (BGS) continues to grow while allograft bone grafts steadily decrease. Synthetic BGS are low in mechanical strength and bioactivity, inspiring the development of novel grafting materials, a traditionally laborious and expensive process. Here a novel BGS derived from sustainably grown coral was evaluated. Coral-derived scaffolds are a natural calcium carbonate bio-ceramic, which induces osteogenesis in bone marrow mesenchymal stem cells (MSCs), the cells responsible for maintaining bone homeostasis and orchestrating fracture repair. By 3D printing MSCs in coral-laden bioinks we utilise high throughput (HT) fabrication and evaluation of osteogenesis, overcoming the limitations of traditional screening methods. MSC and coral-laden GelXA (CELLINK) bioinks were 3D printed in square bottom 96 well plates using a CELLINK BIO X printer with pneumatic adapter Samples were non-destructively monitored during the culture period, evaluating both the sample and the culture media for metabolism (PrestoBlue), cytotoxicity (lactose dehydrogenase (LDH)) and osteogenic differentiation (alkaline phosphatase (ALP)). Endpoint, destructive assays used included qRT-PCR and SEM imaging. The inclusion of coral in the printed bioink was biocompatable with the MSCs, as reflected by maintained metabolism and low LDH release. The inclusion of coral induced osteogenic differentiation in the MSCs as seen by ALP secretion and increased RUNX2, collagen I and osteocalcin transcription. Sustainably grown coral was successfully incorporated into bioinks, reproducibly 3D printed, non-destructively monitored throughout culture and induced osteogenic differentiation in MSCs. This HT fabrication and monitoring workflow offers a faster, less labour-intensive system for the translation of bone substitute materials to clinic. Acknowledgements: This work was co-funded by Enterprise Ireland and Zoan Biomed through Innovation Partnership IP20221024

Traumatic acute or chronic tendon injuries are a wide clinical problem in modern society, resulting in important economic burden to the health system and poor quality of life in patients. Due to the low cellularity and vascularity of tendon tissue the repair process is slow and inefficient, resulting in mechanically, structurally, and functionally inferior tissue. Tissue engineering and regenerative medicine are promising alternatives to the natural healing process for tendon repair, especially in the reconstruction of large damaged tissues. The aim of TRITONE project is to develop a smart, bioactive implantable 3D printed scaffold, able to reproduce the structural and functional properties of human tendon, using FDA approved materials and starting from MSC and their precursor, MPC cell mixtures from human donors. Total cohort selected in the last 12 months was divided in group 1 (N=20) of subjects with tendon injury and group 2 (N=20) of healthy subject. Groups were profiled and age and gender matched. Inclusion criteria were age>18 years and presence of informed consent. Ongoing pregnancy, antihypertensive treatment, cardiovascular diseases, ongoing treatment with anti-aggregants, acetylsalicylic-acid or lithium and age<18 years were exclusion criteria. Firstly, we defined clinical, biological, nutritional life style and genetic profile of the cohort. The deficiency of certain nutrients and sex hormonal differences were correlated with tendon-injured patients. It was established the optimal amount of MPC/MSC human cell (collected from different patients during femoral neck osteotomy). Finally, most suitable biomaterials for tendon regeneration and polymer tendon-like structure were identified. Hyaluronic acid, chemical surface and soft-molecular imprinting (SOFT-MI) was used to functionalize the scaffold. These preliminary results are promising. It will be necessary to enroll many more patients to identify genetic status connected with the onset of tendinopathy. The functional and structural characterization of smart bioactive tendon in dynamic environment will represent the next project step

Adherent cells are known to respond to physical characteristics of their surrounding microenvironment, adapting their cytoskeleton and initiating signaling cascades specific to the type of cue encountered. Scaffolds mimicking native biophysical cues have proven to differentiate stem cells towards tissue-specific lineages and to maintain the phenotype of somatic cells for longer periods of culture time. Although the characteristic anisotropy of tendon tissue is commonly replicated in scaffolds, relevant physical cues such as tendon rigidity or mechanical loading are often neglected. The objective of this study is to use tendons' main extracellular matrix component, collagen type I, to create scaffolds with an anisotropic surface topography and controlled rigidity, in an effort to engineer functional tendon tissue equivalents, with native organization and strength. Porcine collagen type I in solution was treated with one of the following cross-linkers: glutaraldehyde, genipin or 4-arm polyethylene glycol (4SP). The resulting mixture was poured on micro-grooved (2×2×2 μm) or planar polydimethylsiloxane (PDMS) molds and dried in a laminar flow hood to obtain 5 mg/ml collagen films. Surface topography and elastic modulus of the final scaffolds were analyzed using SEM/AFM and rheometry, respectively. Human tendon cells were isolated from adult tendon tissue and cultured on micro-grooved/planar scaffolds for 4, 7 and 10 days. Cell morphology, collagen III and tenascin C expression were analyzed by immunocytochemistry. Among the different cross-linkers used, only the treatment with 4SP resulted in scaffolds with a recognizable micro-grooved surface topography. Precise control over the micro-grooved topography and the rigidity of the scaffolds was achieved by cross-linking the collagen with varying concentrations of 4SP at low pH and temperature. The elastic modulus of the scaffolds cross-linked with the highest concentration of 4SP matched the physiological values reported in developing tendons (∼15 kPa). Around eighty percent of the human tendon cells cultured on the cross-linked collagen films aligned in the direction of the anisotropy for 10 days in culture. At 4 days, tenoyctes cultured on micro-grooved substrates presented a significant higher nuclei aspect ratio than tenocytes cultured on planar substrates for all the 4SP concentrations. Synthesis, deposition and alignment of collagen III and tenascin C, two important tenogenic markers, were up regulated selectively in the rigid micro-grooved scaffolds after 7 days in culture. These results highlight the synergistic effect of matrix rigidity and cell alignment on tenogenic cell lineage commitment. Collectively, this study provides new insights into how collagen can be modulated to create scaffolds with precise imprinted topographies and controlled rigidities. Gene expression analysis and a replicate study with hBMSCs will be carried out to support the first results and to further identify the optimal biophysical conditions for tenogenic cell lineage commitment. This potentially leads to the design of smart implants that not only restore immediate tendon functionality but also provide microscopic cues that drive cellular synthesis of organized tissue-specific matrix

Scaffold-based bone tissue engineering holds great promise for the future of osseous defects therapies. Prepare the suitable scaffold properties are physiochemical modifications in terms of porosity, mechanical strength, cell adhesion, biocompatibility, cell proliferation, mineralization and osteogenic differentiation are required. We produce various bone tissue scaffolds with different techniques such as lyophilization, 3D printing and electrospinning. We wish to overview all the different novel scaffold methods and materials. To improve scaffolds poor mechanical properties, while preserving the porous structure, it is possible to coat the scaffold with synthetic or natural polymers. An increasing interest in developing materials in bone tissue engineering is directed to the organic/inorganic composites that mimic natural bone. Specifically, bone tissue is a composite of an organic and inorganic matrix. Using PLLA, loofah, chitin and cellulose biomaterials we produced bone tissue scaffold with lyophilization technique. Also, using fish scale powder and wet electrospun Poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) a sponge structure had created. Using MRI image data and 3D printer method, a bone tissue scaffold is created by PLA filament. Their mechanical properties had analysed with compression tests and their biocompatibilities had investigated. In order to provide novel strategies for future treatment of bone tumours, the properties of the scaffold, including its in vitro extended-release properties, the inhibition effects of chemotherapeutic agent on the bone tumours and its bone repair capacities were investigated in vitro by using MG63 cells. To develop chemotherapeutic agent-encapsulated poly(lactic-co-glycolic acid) (PLGA) nanoparticles in a porous nano-hydroxyapatite scaffold we aimed to use double emulsion method

Treatment of bone infection often includes a burdensome two-stage revision. After debridement, contaminated implants are removed and replaced with a non-absorbable cement spacer loaded with antibiotics. Weeks later, the spacer is exchanged with a bone graft aiding bone healing. However, even with this two-stage approach infection persists. In this study, we investigated whether a novel 3D-printed, antibiotic-loaded, osteoinductive calcium phosphate scaffold (CPS) is effective in single-stage revision of an infected non-union with segmental bone loss in rabbits. A 5 mm defect was created in the radius of female New Zealand White rabbits. The bone fragment was replaced, stabilized with cerclage wire and inoculated with Staphylococcus aureus (MSSA). After 4 weeks, the infected bone fragment was removed, the site debrided and a spacer implanted. Depending on group allocation, rabbits received: 1) PMMA spacer with gentamycin; 2) CPS loaded with rifampin and vancomycin and 3) Non-loaded CPS. These groups received systemic cefazolin for 4 weeks after revision. Group 4 received a loaded CPS without any adjunctive systemic therapy (n=12 group1-3, n=11 group 4). All animals were euthanized 8 weeks after revision and assessed by quantitative bacteriology or histology. Covariance analysis (ANCOVA) and multiple regression were performed. All animals were culture positive at revision surgery. Half of the animals in all groups had eliminated the infection by end of study. In a historical control group with empty defect and no systemic antibiotic treatment, all animals were infected at euthanasia. There was no significant difference in CFU counts between groups at euthanasia. Our results show that treating an osteomyelitis with segmental bone loss either with CPS or PMMA has a similar cure rate of infection. However, by not requiring a second surgery, the use of CPS may offer advantages over non-resorbable equivalents such as PMMA

Decellularised extracellular matrix scaffolds show great promise for the regeneration of damaged musculoskeletal tissues (cartilage, ligament, meniscus), however, adequate fixation into the joint remains a challenge. Here, we assess the osseo-integration of decellularised porcine bone in a sheep model. This proof-of-concept study supports the overall objective to create composite decellularised tissue scaffolds with bony attachment sites to enable superior fixation and regeneration. Porcine trabecular bone plugs (6mm diameter, 10mm long) were decellularised using a novel bioprocess incorporating low-concentration sodium dodecyl sulphate with protease inhibitors. Decellularised bone scaffolds (n=6) and ovine allograft controls (n=6) were implanted into the condyle of skeletally mature sheep for 4 and 12 weeks. New bone growth was visualised by oxytetracycline fluorescence and standard resin semi-quantitative histopathology. Scaffolds were found to be fully decellularised and maintained the native microarchitecture. Following 4-week implantation in sheep, both scaffold and allograft appeared well integrated. The trabecular spaces of the scaffold were filled with a fibro-mesenchymal infiltrate, but some areas showed a marked focal lymphocytic response, associated with reduced bone deposition. A lesser lymphocytic response was observed in the allograft control. After 12-weeks the lymphocytic reaction was minimised in the scaffold and absent in allografts. The scaffold showed a higher density of new mineralized bone deposition compared to allograft. New marrow had formed in both the scaffold and allografts. Following the demonstration of osteointegration this bioprocess can now be transferred to develop decellularised composite musculoskeletal tissue scaffolds and decellularised bone scaffolds for clinical regeneration of musculoskeletal tissues

The retear of the rotator cuff (RC) repair is a significant problem. Usually it is the effect of poor quality of the tendon. The aim was to evaluate histologically two types of RC reconstruction with scaffold. We have chosen commercially available scaffold polycaprolactone based poly(urethane urea). Rat model of supraspinatus tendon injury was chosen. There were four study groups: RC tear (no repair) (n=10), RC repair (n=10), RC repair augmented with scaffold (n=10) and RC reconstruction with scaffold interposition between tendon and bone (n=10). The repairs were investigated histologically at 6 and 16 weeks. The results in two groups in which scaffold was used had significantly better scores at 6 weeks comparing to non-scaffold groups (16,4±3, 17,3± 2,8 vs. 12,5±4,4, 13,8±1,4 respectively) and 16 weeks (23±1,9, 22,8±1,6 vs. 13,8±3,3, 14,9± 3,8 respectively). Results in two scaffold groups improved between 6 and 16 weeks. Signs of foreign body reaction against scaffold were not observed. Application of scaffold to strengthen the repair site and bridging of the tendon defect improved healing of the RC repair in animal model at 6 and 16 weeks. The quality of reconstructed tendon improved over time. No such effect was observed in groups without repairs and isolated repairs were performed