Restoration a joint's articular surface following degenerative or traumatic pathology to the osteochondral unit pose a significant challenge. Recent advances have shown the utility of collagen-based scaffolds in the regeneration of osteochondral tissue. To provide these collagen scaffolds with the appropriate superstructure novel techniques in 3D printing have been investigated. This study investigates the use of polyɛ-caprolactone (PCL) collagen scaffolds in a porcine cadaveric model to establish the stability of the biomaterial once implanted. This study was performed in a porcine cadaveric knee model. 8mm defects were created in the medial femoral trochlea and repaired with a PCL collagen scaffold.
Purpose. Traumatic articular cartilage (AC) defects are common in young adults and frequently progresses to osteoarthritis. Matrix-Induced Autologous Chondrocyte Implantation (MACI) is a recent advancement in cartilage resurfacing techniques and is a variant of ACI, which is considered by some surgeons to be the gold standard in AC regeneration. MACI involves embedding cultured chondrocytes into a scaffold that is then surgically implanted into an AC defect. Unfortunately, chondrocytes cultured in a normoxic environment (conventional technique) tend to de-differentiate resulting in decreased collagen II and increased collagen I producing in a fibrocartilagous repair tissue that is biomechanically inferior to AC and incapable of withstanding physiologic loads over prolonged periods. The optimum conditions for maintenance of chondrocyte phenotype remain elusive. Normal oxygen tension within AC is <7%. We hypothesized that hypoxic conditions would induce gene expression and matrix production that more closely characterizes normal articular chondrocytes than that achieved under normoxic conditions when chondrocytes are cultured in a collagen scaffold. Method. Chondrocytes were isolated from Outerbridge grade 0 and 1 AC from four patients undergoing total knee arthroplasty and embedded within 216 bovine collagen I scaffolds.
Introduction. Diaphyseal bone defect represents a significant problem for orthopaedic surgeons and patients. Bone is a complex tissue whose structure and function depend strictly on ultrastructural organization of its components: cells, organic (extracellular matrix, ECM) and inorganic components. The purpose of this study was to evaluate bone regeneration in a critical diaphyseal defect treated by implantation of a magnetic scaffold fixed by hybrid system (magnetic and mechanical), supplied through nanoparticle-magnetic (MNP) functionalized with Vascular Endothelial-Growth-Factor-(VEGF) and magnetic-guiding. Methods. A critical long bone defect was created in 8 sheep metatarsus diaphysis: it was 20.0 mm in length; the medullary canal was reamed till 8.00 mm of inner diameter. Then a 8.00 mm diameter magnetic rod was fitted into proximal medullary canal (10 mm in length). After that a scaffold made of Hydroxyapatite (outer diameter 17.00 mm) that incorporates magnetite (HA/Mgn 90/10) was implanted to fill critical long bone defect. A magnetic rod (6.00 mm diameter) was firmly incorporated at proximal side into the scaffold. Both magnets had 10 mm length. To give stability to the complex bone-scaffold-bone a plate was used as a bridge; it was fixed proximally by 2 screws and distally by 3 screws.
The spine is one of the most common sites of bony metastasis, with 80% of prostate, lung, and breast cancers metastasizing to the vertebrae resulting in significant morbidity. Current treatment modalities are systemic chemotherapy, such as Doxorubicin (Dox), administered after resection to prevent cancer recurrence, and systemic antiresorptive medication, such as Zolendronate (Zol), to prevent tumor-induced bone destruction. The large systemic doses required to elicit an adequate effect in the spine often leads to significant side-effects by both drugs, limiting their prolonged use and effectiveness. Recently published work by our lab has shown that biocompatible 3D-printed porous polymer scaffolds are an effective way of delivering Dox locally over a sustained period while inhibiting tumor growth in vitro. Our lab has also generated promising results regarding antitumor properties of Zol in vitro. We aim to develop 3D-printed scaffolds to deliver a combination of Zol and Dox that can potentially allow for a synergistic antitumor activity while preventing concurrent bone loss locally at the site of a tumor, avoiding long systemic exposure to these drugs and decreasing side effects in the clinical setting. The PORO Lay polymer filaments are 3D-printed into 5mm diameter disks, washed with deionized water and loaded with Dox or Zol in aqueous buffer over 7 days. Dox or Zol-containing supernatant was collected daily and the drug release was analyzed over time in a fluorescence plate reader. The polymer-drug (Dox or Zol) release was tested in vitro on prostate and lung cancer cell lines and on prostate- or lung-induced bone metastases cells. Alternatively, direct drug treatment was also carried out on the same cells in vitro. Following treatment, all cells were subject to proliferation assay (MTT and alamar blue), viability assay (LIVE/DEAD), migration assay (Boyden chamber) and invasion assay (3D gel matrix). 3D-printed scaffolds loaded with both Dox and Zol will also be tested on cells. We have established an effective dose (EC50) for prostate and lung cancer cell lines and bone metastases cells with direct treatment with Zol or Dox. We have titrated the drug loading of scaffolds to allow for a release amount of Dox at the EC50 dose over 7 days. In ongoing experiments, we are testing the release of Zol. We have shown Dox releasing scaffolds inhibit cancer cell growth in a 2D culture over 7 days using the above cellular assays and testing the scaffolds with Zol is currently being analyzed. 3D-printed porous polymers like the PORO Lay series of products offer a novel and versatile opportunity for delivery of drugs in future clinical settings. They can decrease systemic exposure of drugs while at the same time concentrating the drugs effect at the site of tumors and consequently inhibit tumor proliferation. Their ability to be loaded with multiple drugs can allow for achieving multiple goals while taking advantage of synergistic effects of different drugs. The ability to 3D-print these polymers can allow for production of custom implants that offer better structural support for bone growth.
Developing biomaterials for bone regeneration that are highly bioactive, resorbable and mechanically strong remains a challenge. Zreiqat's lab recently developed novel scaffolds through the controlled substitution of strontium (Sr) and zinc (Zn) into calcium silicate, to form Sr-Hardystonite and Hardystonite, respectively and investigated their in vivo biocompatibility and osteoconductivity We synthesized 3D scaffolds of Sr-Hardystonite, Hardystonite and compared them to the clinically used tricalcium phosphate (micro-TCP) (6 × 6 × 6 mm) using a polyurethane foam template to produce a porous scaffold. The scaffolds were surgically implanted in the proximal tibial metaphysis of each tibia of Female Wistar rats. Animals were sacrificed at three weeks and six weeks post-implantation and bone formation and scaffold resorption were assessed by microcomputed tomography (micro-CT) histomorphometry and histology. Histological staining on undecalcified sections included Toluidine blue, tartrate-resistant acid phosphatase (TRAP) and alkaline phosphatase (ALP). The bone formation rate and mineral apposition rate will be determined by analysing the extent and separation of fluorescent markers by fluorescent microscopy micro-CT results revealed higher resorbability of the developed scaffolds (Sr-Hardystonite and Hardystonite) which was more pronounced with the Sr-Hardystonite. Toluidine blue staining revealed that the developed ceramics were well tolerated with no signs of rejection, necrosis, or infection. At three weeks post implantation, apparent bone formation was evident both at the periphery and within the pores of the all the scaffolds tested. Bone filled in the pores of the Sr- Hardystonite and Hardystonite scaffolds and was in close contact with the ceramic. In contrast, the control scaffolds showed more limited bone ingrowth and a cellular layer separating the ceramic scaffolds from the bone. By six weeks the Hardystonite and Sr Hardystonite scaffolds were integrated with the bone with most pores filled with new bone. The control scaffold showed new bone formation in the plane of the cortical bone but little new bone where the scaffold entered the marrow space. Sr Hardystonite showed the greatest resorbability with replacement of the ceramic material by bone. We have developed novel engineered scaffolds (Sr-Hardystonite) for bone tissue regeneration. The developed scaffolds resorbed faster than the clinically used micro- TCP with greater amount of bone formation replacing the resorbed scaffold.
Excellent reconstruction of bone will be described induced by a synthetic biomaterial without a calcium phosphate mineral phase or growth factors, and with a pore size of 35 m. The material is fabricated by a process called sphere-templating and it can be made from many synthetic materials including hydrogels, silicones, polyurethanes and glasses. All pores are identical in size and interconnected. Studies from our group have shown optimal healing in soft tissue (as suggested by extensive vascularity and minimal fibrosis) for spherical pores of 30–40 m size. Sphere-templated hydrogel implants in bone were performed using the following procedure: Under appropriate anesthesia, 18–24month old NZW rabbits underwent medial parapatellar arthrotomy, with exposure of the medial femoral condyle. A 3.5 mm end-cutting drill, locked in a rigid armature, was used to create a host graft site at the center of the articular cartilage lesion, with depth of cut matched to the sphere-templated construct thickness of 2 mm. Animals were sacrificed at one day, 28 days, and 12 weeks. After sacrifice, the femora were isolated and the condyles dissected. Condyles were fixed in 4% paraformaldehyde at 4°C for 48 hrs, decalcified in Immunocal for 14 days at 4°C and paraffin embedded. Specimens were sectioned to a thickness and stained with Safranin- O/Fast Green, hematoxylin/eosin or Masson's trichrome. Prior to decalcification, selected samples were evaluated by micro-CT utilising a Skyscan 1076 microCT low dose in-vivo X-ray scanner, slice imaging and 3D image reconstruction. Both histologically, and with micro-CT imaging, excellent tissue and mineral reconstruction was observed in the sphere templated material. The contralateral control, drilled but without implant, showed essentially no reconstruction. Since the classical paradigm for bone reconstruction requires either autologous bone, cadaver bone, or calcium phosphate scaffolds with pores >150 microns, the healing observed here suggests new avenues for bone regeneration.
Traditionally, the gold standard for bone grafting has been either autografts or allografts. Whilst autografts are still widely used, drawbacks such as donor site morbidity are shifting the market rapidly toward the use of orthobiologic bone graft substitutes. This study investigated the in vivo performance of a novel (W02008096334) collagen-hydroxyapatite (CHA) bone graft substitute material as an osteoinductive tissue engineering scaffold. This highly porous CHA scaffold offers significantly increased mechanical strength over collagen-only scaffolds while still exhibiting an extremely high porosity (≈ 99%), and an osteoinductive hydroxyapatite phase [1]. This study assessed the ability of the CHA scaffolds to heal critical-sized (15 mm) long bone segmental defects in vivo, as a viable alternative to autologous bone grafts. Collagen-HA (CHA) composite scaffolds were fabricated based on a previously-described freeze-drying technique [1]. After freeze-drying, these scaffolds were subjected to a dehydrothermal treatment and subsequently chemically crosslinked using EDAC. In vivo performance was assessed using a critical size segmental radial defect (15 mm) introduced into 16 young adult New Zealand White Rabbits under Irish Government license. Animals were divided into three groups; (i) an empty defect group (negative control), (ii) an autogenous bone graft group (positive control) and (iii) a CHA scaffold group (CHA). Segmental defect healing in all animals was assessed using plain X-Ray analysis, at four time-points (0, 6, 12 and 16 weeks). MicroCT and histological analysis were carried out at week 16.Purpose
Method
Autogenous bone grafting limitations have motivated the development of Tissue-Engineered (TE) biomaterials that offer an alternative as bone void fillers. However, the lack of a blood supply within implanted constructs may result in avascular necrosis and construct failure1. The aim of this project was to investigate the potential of novel TE constructs to promote vascularisation and bone defect repair using two distinct approaches. In Study 1, we investigated the potential of a mesenchymal stem cell (MSC) and endothelial cell (EC) co-culture to stimulate pre-vascularisation of biomaterials prior to in vivo implantation2. In Study 2, we investigated the potential of TE hypertrophic cartilage to promote the release of angiogenic factors such as VEGF, vascular invasion and subsequent endochondral bone formation in an in vivo model. Collagen-only (Coll), collagen-glycosaminoglycan (CG) and collagen-hydroxyapatite (CHA) scaffolds were fabricated by freeze-drying3, seeded with cells and implanted into critical-sized calvarial and femoral defects in immunocompetent rats. In Study 1, Coll and CG scaffolds were initially seeded with ECs, allowed to form capillary-like networks before the delayed addition of MSCs and continued culture prior to calvarial implantation. In Study 2, CG and CHA scaffolds were seeded with MSCs and cultured under chondrogenic and subsequent hypertrophic conditions to form a cartilage pre-cursor prior to calvarial and femoral implantation in vivo. MicroCT and histomorphometry quantification demonstrated the ability of both systems to support increased bone formation compared to controls. Moreover, the greatest levels of bone formation were observed in the CG groups, notably in those containing cartilage tissue (Study 2). Assessment of the immune response suggests the addition of MSCs promotes the polarisation of macrophages away from inflammation (M1) towards a pro-remodelling phenotype (M2). We have developed distinct collagen-based systems that promote vascularisation and ultimately enhance bone formation, confirming their potential as advanced strategies for bone repair applications.
Gene-activated scaffolds have shown potential in localised gene delivery resulting in bone tissue regeneration. In this study, the ability of two gene delivery vectors, polyethyleneimine (PEI) and nano-hydroxyapatite (nHA), to act as carriers for the delivery of therapeutic genes when combined with our collagen-nHA (coll-nHA) scaffolds to produce gene-activated scaffolds [1, 2], was determined. In addition, coll-nHA-dual gene scaffolds containing both an angiogenic gene and an osteogenic gene were assessed for bone healing in an
Trufit resorbable scaffolds, made of semiporous copolymer, are press-fit introduced in chondral defects of articular surfaces in order to promote filling and regeneration of damaged bone and cartilage tissues. In another previous work, we have presented our good and promising results obtanied at 1 yy follow-up. Then, we have had the chance to go on on follow-ups and check patients through second-look arthroscopies and serial MRI's: IKDC score showed 38 points improvement. WOMAC score showed statistically significant pain improvement in 89% of cases and function improvement in 86% of cases. Serial MRI's of the knees showed progressive incorporation of the synthetic plugs and no adverse inflammatory reaction. Second-look arthroscopies showed complete and flush fill of the defects and their resurfacing with hyaline-like tissue under different stages of maturation. Recently, we have been able to check, clinically and by serial MRI's, the first patients operated 24 months ago. Despite the mantainance of clinical very good results, as showed by other authors, MRI images showed a delayed biologic process of incorporation of the plugs. This finding has not to be misinterpreted as an implant failure and the post-op rehabilitation has to be continued in order to give regenerating cartilage time to complete the maturation process.
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.
The treatment of acute full thickness chondral damage within the knee is a surgical challenge. Frequently used surgical techniques include chondroplasty, micro-fracture and chondrocyte implantation. These procedures give unpredictable functional outcomes and if the formation of neocartilage is achieved it is predominantly composed of type 1 collagen. The TruFit osteochondral plug was designed to provide a scaffold for cell proliferation into full thickness chondral defects. It is a composite polymer composed of polylactide co-glycolide, calcium sulphate and poly-glycolide fibres. It is composed of 2 layers, one with a similar trabecular network to cancellous bone and a superficial layer designed to simulate articular lining. The TruFit bone plug was analysed using micro-computed tomography. Its morphology characteristics, granulometry, mechanical performance and image guided failure were tested as well as numerical modelling to assess the permeability of TruFit. Morphological parameters of the TruFit bone plug compared favourably with those of human tissue. Under load the scaffold exhibited shear bands throughout the composite leading to a failure mechanism similar to cancellous bone. Stress relaxation rates of the scaffolds were greatly decreased under wet conditions, likely due to plasticisation of the scaffold by water. The biomechanical properties of the TruFit bone plugs are a cause for concern. The