Collagen-hydroxyapatite Scaffold Research Articles

Antibiotic-eluting scaffolds with responsive dual-release kinetics facilitate bone healing and eliminate S. aureus infection

Osteomyelitis (OM) is a progressive, inflammatory infection of bone caused predominately by Staphylococcus aureus. Herein, we engineered an antibiotic-eluting collagen-hydroxyapatite scaffold capable of eliminating infection and facilitating bone healing. An iterative freeze-drying and chemical crosslinking approach was leveraged to modify antibiotic release kinetics, resulting in a layered dual-release system whereby an initial rapid release of antibiotic to clear infection was followed by a sustained controlled release to prevent reoccurrence of infection. We observed that the presence of microbial collagenase accelerated antibiotic release from the crosslinked layer of the scaffold, indicating that the material is responsive to microbial activity. As exemplar drugs, vancomycin and gentamicin-eluting scaffolds were demonstrated to be bactericidal, and supported osteogenesis in vitro. In a pilot murine model of OM, vancomycin-eluting scaffolds were observed to reduce S. aureus infection within the tibia. Finally, in a rabbit model of chronic OM, gentamicin-eluting scaffolds both facilitated radial bone defect healing and eliminated S. aureus infection. These results show that antibiotic-eluting collagen-hydroxyapatite scaffolds are a one-stage therapy for OM, which when implanted into infected bone defects simultaneously eradicate infection and facilitate bone tissue healing.

Healing of large periapical lesion by Regenerative Endodontic procedure in immature and mature permanent teeth with/without collagen-hydroxyapatite scaffold: A Case report

Healing of large periapical lesion by Regenerative Endodontic procedure in immature and mature permanent teeth with/without collagen-hydroxyapatite scaffold: A Case report

Wood-inspired highly anisotropic scaffold activated with in situ mineralized collagen for advanced bone regeneration

Addressing bone defects, limited by inherent regenerative capacities, has catalyzed the development of biomimetic scaffolds. Despite the extensive exploration of collagen-hydroxyapatite scaffolds, they frequently fail to accurately replicate the intricate nanostructure of natural bone. We introduce the wood-collagen- hydroxyapatite (WCH) scaffold, a novel biomimetic structure inspired by the analogous architectures of wood and bone, aimed at substantially improving bone regeneration. Comprising a wood-based matrix, collagen interface, and hydroxyapatite mineralization, the WCH scaffold embodies the critical components of bone tissue. The scaffold’s wood-derived framework imparts an anisotropic structure and notable mechanical strength, closely mimicking natural bone’s nanoarchitecture. Significantly, the scaffold’s longitudinal (L) orientation outperforms its radial counterpart in biocompatibility and bioactivity, reflecting bone’s directional properties and promoting superior cell proliferation, adhesion, and osteogenic differentiation. Validated in a rabbit femur defect model, the L directional WCH scaffold significantly accelerated new bone formation, tissue ingrowth, and bone tissue maturation. This innovative wood-inspired scaffold, characterized by its highly anisotropic structure and enhanced with in-situ mineralized collagen, holds significant promise for advancing bone regeneration techniques.

A Biomimetic Osteochondral Scaffold Augmented With Filtered Bone Marrow Aspirate for the Treatment of Joint Surface Lesions in the Knee

Background: Multilayered osteochondral scaffolds are becoming increasingly utilized for the repair of knee joint surface lesions (KJSLs). However, the literature on predictive factors is rather limited. Purpose: To (1) evaluate the clinical outcomes and safety of a combined single-step approach using a biomimetic collagen-hydroxyapatite scaffold (CHAS) and filtered bone marrow aspirate (fBMA) for the treatment of KJSLs and (2) identify significant predictors of the treatment outcomes. Study Design: Case series; Level of evidence, 4. Methods: All patients who underwent surgery because of a KJSL (size ≥1.5 cm2; International Cartilage Regeneration & Joint Preservation Society grades 3-4) using the combination above were selected from a hospital registry database (100 patients; minimum 2-year follow-up). Patient characteristics, medical history, knee joint and lesion status, intraoperative details, and cellular parameters of the injected fBMA were collected. The arthroscopic evaluation of chondral and meniscal tissue quality in all knee compartments was performed using the Chondropenia Severity Score. Treatment outcomes were determined clinically using patient-reported outcome measures (Knee Injury and Osteoarthritis Outcome Score, EuroQol–5 Dimensions–3 Levels, EuroQol–Visual Analog Scale, and Tegner Activity Scale) and by assessing the occurrence of serious adverse events and graft failure. Multivariable regression analysis was performed to identify significant predictors of the treatment outcomes. Results: At a mean follow-up of 54.2 ± 19.4 months, 78 (87%) patients completed the questionnaires with significant improvements toward the baseline (P < .00625): KOOS Pain subscale from 62 ± 17 to 79 ± 18, KOOS Total score from 57 ± 16 to 70 ± 20, EuroQol–Visual Analog Scale from 61 ± 21 to 76 ± 16, EuroQol–5 Dimensions–3 Levels from 0.57 ± 0.20 to 0.80 ± 0.21, and Tegner Activity Scale from 2.8 ± 1.5 to 3.9 ± 1.9. The graft failure rate was 4%. A longer duration of preoperative symptoms, previous surgery, larger lesions, older age, and female sex were the main negative predictors for the treatment outcomes. The Chondropenia Severity Score and the number of fibroblast colony-forming units in fBMA positively influenced some of the clinical results and safety. Conclusion: A CHAS augmented with fBMA proved to be an adequate and safe approach for the treatment of KJSLs up to midterm follow-up. Based on the subanalysis of predictive factors, the surgical intervention should be performed in a timely and precise manner to prevent lesion enlargement, deterioration of the general knee cartilage status, and recurrent surgical procedures, especially in older and female patients. When a CHAS is used, the quantity of MSCs seems to play a role in augmentation. Registration: NCT06078072 (ClinicalTrials.gov identifier).

Immunomodulation Using BMP-7 and IL-10 to Enhance the Mineralization Capacity of Bone Progenitor Cells in a Fracture Hematoma-Like Environment.

Following biomaterial implantation, a failure to resolve inflammation during the formation of a fracture hematoma can significantly limit the biomaterial's ability to facilitate bone regeneration. This study aims to combine the immunomodulatory and osteogenic effects of BMP-7 and IL-10 with the regenerative capacity of collagen-hydroxyapatite (CHA) scaffolds to enhance in vitro mineralization in a hematoma-like environment. Incubation of CHA scaffolds with human whole blood leads to rapid adsorption of fibrinogen, significant stiffening of the scaffold, and the formation of a hematoma-like environment characterized by a limited capacity to support the infiltration of human bone progenitor cells, a significant upregulation of inflammatory cytokines and acute phase proteins, and significantly reduced osteoconductivity. CHA scaffolds functionalized with BMP-7 and IL-10 significantly downregulate the production of key inflammatory cytokines, including IL-6, IL-8, and leptin, creating a more permissive environment for mineralization, ultimately enhancing the biomaterial's osteoconductivity. In conclusion, targeting the onset of inflammation in the early phase of bone healing using BMP-7 and IL-10 functionalized CHA scaffolds is a promising approach to effectively downregulate inflammatory processes, while fostering a more permissive environment for bone regeneration.

Nile Tilapia Skin-Derived Materials for Dental Application

The skin of Nile tilopia has gained attention in biomaterials science because of its low cost, abundance, and ability to recover from waste products of the fish industry. Nile tilapia skin can yield around 40% dry weight of type I highly biocompatible collagen. Nile tilopia collagen is used successfully for wound healing and hemostatic dressing. Collagen membranes and collagen-hydroxyapatite scaffolds derived from Nile tilapia skin are effectively employed for guided tissue regeneration. Future research is crucial to overcome limitations such as weak mechanical strength and cell compatibility.

TRANSFECTION OF HMSCS WITH CHEMICALLY MODIFIED MRNA CODING FOR BMP-7 ENHANCES OSTEOGENESIS

Bone morphogenetic proteins (BMPs) have been widely investigated for treating non-healing fractures. They participate in bone reconstruction by inducing osteoblast differentiation, and osteoid matrix production.1 The human recombinant protein of BMP-7 was among the first growth factors approved for clinical use. Despite achieving comparable results to autologous bone grafting, severe side effects have been associated with its use.2 Furthermore, BMP-7 was removed from the market.3 These complications are related to the high doses used (1.5-40 miligrams per surgery)2 compared to the physiological concentration of BMP in fracture healing (in the nanogram to picogram per milliliter range).4 In this study, we use transcript therapy to deliver chemically modified mRNA (cmRNA) encoding BMP-7. Compared to direct use of proteins, transcript therapy allows the sustained synthesis of proteins with native conformation and true post-translational modifications using doses comparable to the physiological ones.5 Moreover, cmRNA technology overcomes the safety and affordability limitations of standard gene therapy i.e. pDNA.6 BMP-7 cmRNA was delivered using Lipofectamine™ MessengerMAX™ to human mesenchymal stromal cells (hMSCs). We assessed protein expression and osteogenic capacity of hMSCs in monolayer culture and in a house-made, collagen hydroxyapatite scaffold. Using fluorescently-labelled cmRNA we observed an even distribution after loading complexes into the scaffold and a complete release after 3 days. For both monolayer and 3D culture, BMP-7 production peaked at 24 hours post-transfection, however cells transfected in scaffolds showed a sustained expression. BMP-7 transfected hMSCs yielded significantly higher ALP activity and Alizarin red staining at later timepoints compared to the untransfected group. Interestingly, BMP-7 cmRNA treatment triggered expression of osteogenic genes like OSX, RUNX-2 and OPN, which was also reflected in immunostainings. This work highlights the relevance of cmRNA technology that may overcome the shortcomings of protein delivery while circumventing issues of traditional pDNA-based gene therapy for bone regeneration.Acknowledgement: This work has been performed as part of the cmRNAbone project and has received funding from the European Union's Horizon 2020 research and innovation programme under the Grant Agreement No 874790.

Collagen-Hydroxyapatite Scaffold Platform Incorporating Dimethyloxalylglycine Loaded Polymersomes Decorated with BMP-2 Peptide for Bone Tissue Engineering

Introduction: Recent advances in the field of nanotechnology have led to the development of platforms for healing or repairing damaged tissues. The present study aimed to provide a bone extracellular matrix (ECM) mimetic platform made of hydroxyapatite and collagen loaded with nanopolymersomes encapsulating dimethyloxalylglycine (DMOG) and decorated with BMP-2 peptide in order to accelerate and increase the differentiation of mesenchymal stem cells to bone tissue. Method: In this study, polymersome nanoparticles carrying DMOG and targeted with BMP-2 peptide were prepared. The physicochemical properties of nanoparticles were evaluated. Thereafter, it was loaded into collagen-nanohydroxyapatite scaffolds and the expression levels of osteogenic genes OCN and OPN and angiogenic genes vcam, vwf, and kdr were checked in mesenchymal stem cells. Results: The surface charge and particle size of the synthesized nanoparticles were reported as -6 mv and 141 nm, respectively. The nanoparticles were able to slowly release 35% of the loaded DMOG within 30 days. The results demonstrated that the expression levels of OCN and OPN genes in cells exposed to polymersomes targeted with BMP-2 peptide were 27 and 2 times more than those of non-targeted polymersomes, respectively. In addition, the expression of angiogenic genes, vwf, kdr, and vcam, displayed a significant increase in targeted polymersomes compared to the non-targeted types (11.7-, 12.8-, and 2.17-fold, respectively). Conclusion: It seems that providing an environment similar to the bone ECM by combining an osteomimetic scaffold made of collagen-nanohydroxyapatite, DMOG as an angiogenic factor, and BMP-2 as the osteogenic factor can be effective in accelerating and increasing the differentiation of mesenchymal stem cells into osteoblasts.

Sustained delivery of a heterodimer bone morphogenetic protein-2/7 via a collagen hydroxyapatite scaffold accelerates and improves critical femoral defect healing.

Despite the glimmer of hope provided by the discovery and commercialization of bone morphogenetic protein-2 (BMP-2) as a bone graft substitute, side effects related to the use of supraphysiological doses have hindered its clinical usage. In this study, we compared the osteoinductive potential of BMP-2 homodimer with a heterodimer of BMP-2/7, both delivered via a collagen-hydroxyapatite (CHA) scaffold delivery system, with the aim to reduce the overall therapeutic BMP doses and the associated side-effects. We first show that the incorporation of hydroxyapatite in collagen-based BMP delivery systems is pivotal for achieving efficient BMP sequestration and controlled release. Using an ectopic implantation model, we then showed that the CHA+BMP-2/7 was more osteoinductive than CHA+BMP-2. Further evaluation of the molecular mechanisms responsible for this increased osteoinductivity at an early stage in the regeneration process indicated that the CHA+BMP-2/7 enhanced progenitor cell homing at the implantation site, upregulated the key transcriptomic determinants of bone formation, and increased the production of bone extracellular matrix components. Using fluorescently labelled BMP-2/7 and BMP-2, we demonstrated that the CHA scaffold provided a long-term delivery of both molecules for at least 20 days. Finally, using a rat femoral defect model, we showed that an ultra-low dose (0.5 µg) of BMP-2/7 accelerated fracture healing and performed at a level comparable to 20-times higher BMP-2 dose. Our results indicate that the sustained delivery of BMP-2/7 via a CHA scaffold could bring us a step closer in the quest for the use of physiological growth factor doses in fracture healing. STATEMENT OF SIGNIFICANCE: • Incorporation of hydroxyapatite (HA) in a collagen scaffold dramatically improves bone morphogenic protein (BMP) sequestration via biophysical interactions with BMP, thereby providing more controlled BMP release compared with pristine collagen. • We then investigate the molecular mechanisms responsible for increased osteoinductive potential of a heterodimer BMP-2/7 with is clinically used counterpart, the BMP-2 homodimer. • The superior osteoinductive properties of BMP-2/7 are a consequence of its direct positive effect on progenitor cell homing at the implantation site, which consequently leads to upregulation of cartilage and bone related genes and biochemical markers. • An ultra-low dose of BMP-2/7 delivered via a collagen-HA (CHA) scaffold leads to accelerated healing of a critical femoral defect in rats while a 20-times higher BMP-2 dose was required to achieve comparable results.

Monitoring of freezing patterns within 3D collagen-hydroxyapatite scaffolds using infrared thermography

The importance of cryopreservation in tissue engineering is unceasingly increasing. Preparation, cryopreservation, and storage of tissue-engineered constructs (TECs) at an on-site location offer a convenient way for their clinical application and commercialization. Partial freezing initiated at high sub-zero temperatures using ice-nucleating agents (INAs) has recently been applied in organ cryopreservation. It is anticipated that this freezing technique may be efficient for the preservation of both scaffold mechanical properties and cell viability of TECs. Infrared thermography is an instrumental method to monitor INAs-mediated freezing of various biological entities.In this paper, porous collagen-hydroxyapatite (collagen-HAP) scaffolds were fabricated and characterized as model TECs, whereas infrared thermography was proposed as a method for monitoring the crystallization-related events on their partial freezing down to −25 °C. Intra- and interscaffold latent heat transmission were descriptively evaluated. Nucleation, freezing points as well as the degree of supercooling and duration of crystallization were calculated based on inspection of respective thermographic curves. Special consideration was given to the cryoprotective agent (CPA) composition (Snomax®, crude leaf homogenate (CLH) from Hippophae rhamnoides, dimethyl sulfoxide (Me2SO) and recombinant type-III antifreeze protein (AFP)) and freezing conditions (‘in air’ or ‘in bulk CPA’).For CPAs without ice nucleation activity, thermographic measurements demonstrated that the supercooling was significantly milder in the case of scaffolds present in a CPA solution compared to that without them. This parameter (ΔT, °C) altered with the following tendency: 10 Me2SO (2.90 ± 0.54 (‘scaffold in a bulk CPA’) vs. 7.71 ± 0.43 (‘bulk CPA’, P < 0.0001)) and recombinant type-III AFP, 0.5 mg/ml (2.65 ± 0.59 (‘scaffold in a bulk CPA’) vs. 7.68 ± 0.34 (‘bulk CPA’, P < 0.0001)).At the same time, in CPA solutions with ice nucleation activity the least degree of supercooling and the longest crystallization duration (Δt, min) for scaffolds frozen ‘in air’ were documented for CLH from Hippophae rhamnoides (1.57 ± 0.37 °C and 21.86 ± 2.93 min) compared to Snomax, 5 μg/ml (2.14 ± 0.33 °C and 19.91 ± 4.72 min), respectively).Moreover, when frozen ‘in air’ in CLH from Hippophae rhamnoides, collagen-HAP scaffolds were shown to have the longest ice-liquid equilibrium phase during crystallization and the lowest degree of supercooling followed by alginate core-shell capsules and nanofibrous electrospun fiber mats made of poly ɛ-caprolactone (PCL) and polylactic acid (PLA) (PCL/PLA) blend.The paper offers evidence that infrared thermography provides insightful information for monitoring partial freezing events in TECs when using different freezing containers, CPAs and conditions. This may further TEC-specific cryopreservation with enhanced batch homogeneity and optimization of CPA compositions of natural origin active at warm sub-zero temperatures.

Interleukin-1 receptor antagonist enhances the therapeutic efficacy of a low dose of rhBMP-2 in a weight-bearing rat femoral defect model

In the clinical treatment of fractures, rhBMP-2 administration is associated with a well-established profile of side-effects, including osteolysis and ectopic bone formation, which are driven by pro-inflammatory processes triggered by the use of high doses. Immunomodulatory strategies could minimize the incidence of side-effects by enabling the use of lower, and safer, rhBMP-2 doses. This study investigated whether interleukin-1 receptor antagonist (IL-1Ra) can enhance the therapeutic efficacy of a low dose of rhBMP-2 in a weight-bearing femoral fracture healing model. Exogenous IL-1Ra, in combination with rhBMP-2, was delivered using a collagen-hydroxyapatite scaffold (CHA) to attenuate IL-1β produced in response to fracture. Femoral defects were treated with CHA scaffolds alone, or loaded with IL-1Ra (2.5 µg), rhBMP-2 (1 µg), IL-1Ra (2.5 µg) in combination with rhBMP-2 (1 µg). Bone healing was assessed over 14 weeks in comparison to control groups, empty defect, and a higher dose of rhBMP-2 (5 µg), which were recently demonstrated to lead to non-union, and successful bridging of the defect, respectively. The combination of IL-1Ra and rhBMP-2 led to significantly faster early bone formation, at both week 4 and 6, compared to a low dose of rhBMP-2 alone. By 14 weeks, the combination of IL-1Ra and a rhBMP-2 promoted full bridging of femurs, which were 3-fold more mechanically reliable compared to the femurs treated with a low dose of rhBMP-2 alone. Taken together, this study demonstrates that IL-1Ra can significantly enhance femoral bone healing when used in combination with a low dose of rhBMP-2. Statement of significanceEnabling the use of lower and safer doses of rhBMP-2, a potent inducer of bone formation, is of clinical relevance in orthopaedic medicine. In this study, the immunomodulatory interleukin-1 receptor antagonist (IL-1Ra) was investigated for its capacity to enhance the therapeutic efficacy of rhBMP-2 when used at lower doses in a weight-bearing femoral fracture healing model. The combination of IL-1Ra and rhBMP-2 led to significantly faster early bone formation, and resulted in more mechanically reliable healed femurs, compared to a low dose of rhBMP-2 alone. This demonstrates for the first time in a rat long bone healing model that IL-1Ra can significantly enhance bone healing when used in combination with a low dose of rhBMP-2.

A novel natural-derived tilapia skin collagen mineralized with hydroxyapatite as a potential bone-grafting scaffold.

Collagen is widely used in medical field because of its excellent biocompatibility and bioactivity. To date, collagen for biomedical use is always derived from bovine or swine. The purpose of this study was to evaluate collagen-based biomaterials from non-mammalian donors for bone repair. Thus, tilapia skin collagen-hydroxyapatite (T-col/HAp) scaffolds were fabricated in three different proportions and then cross-linked with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide-N-hydroxysuccinimide (EDC-NHS). The scaffolds were evaluated for their microstructure, chemical and physical properties, mechanical strength and degradability. Then the in vitro responses of bone mesenchymal stem cells (BMSCs) to the scaffolds were investigated in terms of cellular proliferation, differentiation, and mineralization. At last, the scaffolds were implanted into rat skull critical defections to investigate the potential of osteogenic activities. As a result, the pore sizes and the porosities of the scaffolds were approximately 106.67-196.67μm and 81.5%-66.7%. Pure collagen group showed a mechanical strength of 0.065MPa, and the mechanical strength was significantly enhanced almost 17 times and 32 times in collagen/HAp ratio 1:4 and 1:9 groups. In vitro studies revealed the most prominent and healthy growth of BMSCs in collagen/HAp ratio 1:4 group. All the scaffolds showed certain osteogenic activities and those loaded with small amount of hydroxyapatite showed the strongest bioactivities. The micro-CT showed that the critical bone defect was almost filled with generated bone 6months after implantation in collagen/HAp ratio 1:4 group. The biomechanics tests further confirmed the highest generated bone strength was in the collagen/HAp ratio 1:4 group. This study indicated aquatic collagen might be a potential alternative for type I collagen from mammals in bone tissue engineering. The combination of collagen and inorganic materials was also important and appropriate inorganic component loading can achieve both osteogenic quality and osteogenic efficiency to a certain extent.

A comparative in vitro and in vivo study on bone tissue engineering potential of the collagen/nano-hydroxyapatite scaffolds loaded with ginger extract and curcumin

Tissue engineering, a multidisciplinary science which can be used to restore the functions of the damaged tissues and organs, has attracted attention of researchers for the last decades. In this study, due to the importance of the role of scaffold in tissue engineering, optimized collagen-hydroxyapatite (COL-HA) scaffolds have been loaded with different amounts of curcumin (CUR) and hydro-alcoholic ginger extract (GIN) for bone tissue engineering applications. The scaffolds were created by freeze dryer method and the physical and biological properties of the scaffolds were evaluated in vitro and in vivo. The comparison revealed the efficacy of resulting porous 3D structures in both groups of scaffolds with CUR and GIN. Mechanically, CUR increased the tensile strength of the scaffolds, while GIN slightly decreased it. Both CUR and GIN caused higher bioactivity and osteogenesis ability in the COL-HA scaffolds. In vitro biocompatibility, in the presence of mesenchymal stem cells derived from synovial membrane of rats, has been improved by CUR and GIN. In vivo biocompatibility, by subcutaneous implantation of the scaffold in rat animal models for 12 weeks, indicated less inflammatory reactions by the addition of CUR and GIN into the scaffolds. These findings make promising appropriate scaffolds for bone tissue engineering applications.

Incorporation of hydroxyapatite into collagen scaffolds enhances the therapeutic efficacy of rhBMP-2 in a weight-bearing femoral defect model

Traditional methods to promote bone healing, such as autologous bone grafting, and therapeutic strategies involving recombinant bone morphogenetic proteins (BMPs), such as BMP-2 and BMP-7, have been associated with performance limitations and dose-related safety concerns, respectively. Thus, there is an unmet need for osteoinductive bone graft substitutes that can enhance the therapeutic efficacy of rhBMP-2 and facilitate dose-reduction approaches. This study aimed to determine the therapeutic efficacy of rhBMP-2 delivered using a collagen-only scaffold (CO) (representative of clinical standards), a collagen-hydroxyapatite scaffold (CHA), and a thermoresponsive hyaluronic acid hydrogel doped with gelatin and laminin (HyA-GL). Efficacy over 14 weeks was evaluated using 2 mm segmental femoral defects in skeletally mature female F344 rats, internally fixated using a 1.25 mm-thick polyetheretherketone plate. Empty defects failed to bridge but administration of rhBMP-2 using either CO or CHA scaffolds led to successful bridging, while using HyA-GL resulted in non-union. CHA scaffolds induced enhanced and stiffer new bone formation compared to both CO and HyA-GL scaffolds. Limited luminal remodeling occurred in both CO and CHA groups. Taken together, this study has determined that a collagen-hydroxyapatite scaffold can enhance the therapeutic efficacy of rhBMP-2 compared to collagen-only scaffolds routinely used in standard clinical practice.

Cell-free osteochondral scaffolds provide a substantial clinical benefit in the treatment of osteochondral defects at a minimum follow-up of 5\u2009years

PurposeThe treatment of osteochondral lesions is challenging and no consensus has been established about the best option for restoring both cartilage and subchondral bone. Multilayer collagen-hydroxyapatite scaffolds have shown promising clinical results, but the outcome at a follow-up longer than 5 years still has to be proved. The aim was to evaluate the clinical outcome of patients with a knee isolated osteochondral lesion treated with a biomimetic three-layered scaffold at a minimum 5 years of follow-up.MethodsTwenty-nine patients (23 males and 6 females, mean age 31.5 ± 11.4 years) were evaluated retrospectively before surgery, at 1 and 2 years and at last follow-up (FU). Visual Analog Scale (VAS) for pain, International Knee Documentation Committee (IKDC) Subjective Score, Tegner-Lysholm Knee Scoring Scale and Tegner Activity Level Scale were collected. Mean FU was 7.8 ± 2.0 years (min 5.1 - max 11.3). The etiology of the defect was Osteochondritis Dissecans or osteonecrosis (17 vs 12 cases).ResultsAt 12 months FU the IKDC score improved from 51.1 ± 21.7 to 80.1 ± 17.9 (p < 0.01), Tegner Lysholm Score from 59.9 ± 17.3 to 92.5 ± 9.0 (p < 0.01), VAS from 6.1 ± 2.1 to 1.7 ± 2.3 (p < 0.01) and Tegner Activity Level Scale from 1.6 ± 0.5 to 4.9 ± 1.7 (p < 0.01). The results remained stable at 24 months, while at last FU a statistically significant decrease in IKDC, Tegner Lysholm and Tegner Activity Scale was recorded, though not clinically relevant. Patients under 35 achieved statistically better outcomes.ConclusionsThe use of a cell-free collagen-hydroxyapatite osteochondral scaffold provides substantial clinical benefits in the treatment of knee osteochondral lesions at a minimum follow-up of 5 years, especially in patients younger than 35 years.Level of evidenceLevel IV.

Sequential dual-drug delivery of BMP-2 and alendronate from hydroxyapatite-collagen scaffolds for enhanced bone regeneration

The clinical use of bioactive molecules in bone regeneration has been known to have side effects, which result from uncontrolled and supraphysiological doses. In this study, we demonstrated the synergistic effect of two bioactive molecules, bone morphogenic protein-2 (BMP-2) and alendronate (ALN), by releasing them in a sequential manner. Collagen-hydroxyapatite composite scaffolds functionalized using BMP-2 are loaded with biodegradable microspheres where ALN is encapsulated. The results indicate an initial release of BMP-2 for a few days, followed by the sequential release of ALN after two weeks. The composite scaffolds significantly increase osteogenic activity owing to the synergistic effect of BMP-2 and ALN. Enhanced bone regeneration was identified at eight weeks post-implantation in the rat 8-mm critical-sized defect. Our findings suggest that the sequential delivery of BMP-2 and ALN from the scaffolds results in a synergistic effect on bone regeneration, which is unprecedented. Therefore, such a system exhibits potential for the application of cell-free tissue engineering.

Gene activated scaffolds incorporating star-shaped polypeptide-pDNA nanomedicines accelerate bone tissue regeneration in vivo.

Increasingly, tissue engineering strategies such as the use of biomaterial scaffolds augmented with specific biological cues are being investigated to accelerate the regenerative process. For example, significant clinical challenges still exist in efficiently healing large bone defects which are above a critical size. Herein, we describe a cell-free, biocompatible and bioresorbable scaffold incorporating a novel star-polypeptide biomaterial as a gene vector. This gene-loaded scaffold can accelerate bone tissue repair in vivo in comparison to a scaffold alone at just four weeks post implantation in a critical sized bone defect. This is achieved via the in situ transfection of autologous host cells which migrate into the implanted collagen-based scaffold via gene-loaded, star-shaped poly(l-lysine) polypeptides (star-PLLs). In vitro, we demonstrate that star-PLL nanomaterials designed with 64 short poly(l-lysine) arms can be used to functionalise a range of collagen based scaffolds with a dual therapeutic cargo (pDual) of the bone-morphogenetic protein-2 plasmid (pBMP-2) and vascular endothelial growth factor plasmid (pVEGF). The versatility of this polymeric vector is highlighted in its ability to transfect Mesenchymal Stem Cells (MSCs) with both osteogenic and angiogenic transgenes in a 3D environment from a range of scaffolds with various macromolecular compositions. In vivo, we demonstrate that a bone-mimetic, collagen-hydroxyapatite scaffold functionalized with star-PLLs containing either 32- or 64- poly(l-lysine) arms can be used to successfully deliver this pDual cargo to autologous host cells. At the very early timepoint of just 4 weeks, we demonstrate the 64-star-PLL-pDual functionalised scaffold as a particularly efficient platform to accelerate bone tissue regeneration, with a 6-fold increase in new bone formation compared to a scaffold alone. Overall, this article describes for the first time the incorporation of novel star-polypeptide biomaterials carrying two therapeutic genes into a cell free scaffold which supports accelerated bone tissue formation in vivo.

Hydroxyapatite Particle Shape and Size Influence MSC Osteogenesis by Directing the Macrophage Phenotype in Collagen-Hydroxyapatite Scaffolds.

The field of bone tissue engineering has seen the advancement of a variety of biomaterials with a diverse range of material properties. Biomaterial properties such as particle shape and size, stiffness, and pore size all influence the osteogenic capacity of biomaterials, typically evaluated in vitro by analyzing their potential to promote osteogenesis in mesenchymal stem cells (MSCs). There is now accumulating evidence highlighting the role of macrophages in driving bone regeneration responses. In this study, we evaluated the osteogenic capacity of collagen scaffolds functionalized with hydroxyapatite particles of varying shapes (needle vs spherical) and sizes (5 μm vs 100 μm) using an in vitro culture system of MSCs alone and in coculture with macrophages. We show that macrophage response to HA particles was elevated in the presence of a scaffold with 5 μm needle-shaped particles (Coll N5), with an increase in the expression and secretion of both pro-inflammatory (TNFα, IL6, and MIP1α) and anti-inflammatory (IL10 and IL1Ra) factors. When MSCs alone were cultured on the scaffolds, we show that scaffolds with HA particles were highly osteogenic, with superior osteogenesis observed in scaffolds with large 30 μm spherical particles (Coll S30) compared to small 5 μm needle-shaped particles (Coll N5). A coculture of MSCs with macrophages increased osteogenesis in all groups, with the most dramatic increase on Coll N5 scaffolds, leading to an elimination of the differences observed during monoculture. Through gene expression analysis, we showed that this correlated with an enhanced pro-osteogenic macrophage phenotype on Coll N5 scaffolds. These results highlight the potential of modulating material properties such as particle shape and size to develop osteoimmunomodulatory materials that direct osteogenic responses by influencing macrophage response.

Non-viral Gene Delivery of Interleukin-1 Receptor Antagonist Using Collagen-Hydroxyapatite Scaffold Protects Rat BM-MSCs From IL-1β-Mediated Inhibition of Osteogenesis.

Although most bone fractures typically heal without complications, a small proportion of patients (≤10%) experience delayed healing or potential progression to non-union. Interleukin-1 (IL-1β) plays a crucial role in fracture healing as an early driver of inflammation. However, the effects of IL-1β can impede the healing process if they persist long after the establishment of a fracture hematoma, making it a promising target for novel therapies. Accordingly, the overall objective of this study was to develop a novel gene-based therapy that mitigates the negative effects of IL-1β-driven inflammation while providing a structural template for new bone formation. A collagen-hydroxyapatite scaffold (CHA) was used as a platform for the delivery of nanoparticles composed of pDNA, encoding for IL-1 receptor antagonist (IL-1Ra), complexed to the robust non-viral gene delivery vector, polyethyleneimine (PEI). Utilizing pDNA encoding for Gaussia luciferase and GFP as reporter genes, we found that PEI-pDNA nanoparticles induced a transient gene expression profile in rat bone marrow-derived mesenchymal stromal cells (BM-MSCs), with a transfection efficiency of 14.8 ± 1.8% in 2D. BM-MSC viability was significantly affected by PEI-pDNA nanoparticles as evaluated using CellTiter Blue; however, after 10 days in culture this effect was negligible. Transfection with PEI-pIL-1Ra nanoparticles led to functional IL-1Ra production, capable of antagonizing IL-1β-induced expression of secreted embryonic alkaline phosphatase from HEK-Blue-IL-1β reporter cells. Sustained treatment with IL-1β (0.1, 1, and 10 ng/ml) had a dose-dependent negative effect on BM-MSC osteogenesis, both in terms of gene expression (Alpl and Ibsp) and calcium deposition. BM-MSCs transfected with PEI-IL-1Ra nanoparticles were found to be capable of overcoming the inhibitory effects of sustained IL-1β (1 ng/ml) treatments on in vitro osteogenesis. Ultimately, IL-1Ra gene-activated CHA scaffolds supported mineralization of BM-MSCs under chronic inflammatory conditions in vitro, demonstrating potential for future therapeutic applications in vivo.

Treatment of Juvenile Knee Osteochondritis Dissecans with a Cell-Free Biomimetic Osteochondral Scaffold: Clinical and MRI Results at Mid-Term Follow-up.

Osteochondral surgical procedures have been described for the treatment of unfixable osteochondritis dissecans (OCD), but only few of them have been studied for juvenile OCD (JOCD) lesions. A cell-free biomimetic osteochondral scaffold showed positive results in adult patients. The aim of this study was to evaluate the results of this scaffold for the treatment of knee JOCD at mid-term follow-up. Twenty patients (14 males, 6 females) were included in this study. Mean age was 16.2 ± 1.4 years, average defect size was 3.2 ± 1.8 cm2, and mean symptoms duration was 20.2 ± 17.9 months. After the implantation of the osteochondral collagen-hydroxyapatite scaffold (Maioregen, Fin-Ceramica, Faenza, Italy), patients were evaluated preoperatively and prospectively at 1, 2, and at final mean follow-up of 6 years (range 5-7 years) with International Knee Documentation Committee (IKDC) subjective and objective, Tegner, and EuroQol visual analogue scale (VAS) scores. MRI evaluation was performed with the MOCART 2.0 score. All scores showed a significant improvement. IKDC subjective score went from 50.3 ± 17.4 preoperative score to 75.3 ± 14.6 at 1 year (P = 0.002), 80.8 ± 14.6 at 2 years and 85.0 ± 9.3 at 6 years. The Tegner score improved from the preoperative evaluation of 2.6 ± 1.4 to 5.5 ± 2.0 at 6 years (P < 0.0005), although without reaching the level registered before the onset of symptoms. A longer symptoms duration influenced negatively IKDC subjective and Tegner scores up to 2 years (P = 0.003 and P = 0.002, respectively) but did not affect the final outcome. Lesion size did not affect the final result. The MOCART 2.0 score showed a significant improvement between 1-year and final follow-up, but with persisting subchondral alterations. This study demonstrated a clinical improvement stable over time with a high survival rate, although with persisting abnormal MRI findings, especially at subchondral bone level. This procedure can be considered a suitable option for the treatment of young patients affected by knee OCD. Level of evidence. Case series, level IV.