Characterization of Biocompatibility of Functional Bioinks for 3D Bioprinting.

Three-dimensional (3D) bioprinting with suitable bioinks has become a critical tool for fabricating 3D biomimetic complex structures mimicking physiological functions. While enormous efforts have been devoted to developing functional bioinks for 3D bioprinting, widely accepted bioinks have not yet been developed because they have to fulfill stringent requirements such as biocompatibility and printability simultaneously. To further advance our knowledge of the biocompatibility of bioinks, this review presents the evolving concept of the biocompatibility of bioinks and standardization efforts for biocompatibility characterization. This work also briefly reviews recent methodological advances in image analyses to characterize the biocompatibility of bioinks with regard to cell viability and cell-material interactions within 3D constructs. Finally, this review highlights a number of updated contemporary characterization technologies and future perspectives to further advance our understanding of the biocompatibility of functional bioinks for successful 3D bioprinting.

Effects of surface patterning and topography on the cellular functions of tissue engineered scaffolds with special reference to 3D bioprinting.

The extracellular matrix (ECM) of the tissue organ exhibits a topography from the nano to micrometer range, and the design of scaffolds has been inspired by the host environment. Modern bioprinting aims to replicate the host tissue environment to mimic the native physiological functions. A detailed discussion on the topographical features controlling cell attachment, proliferation, migration, differentiation, and the effect of geometrical design on the wettability and mechanical properties of the scaffold are presented in this review. Moreover, geometrical pattern-mediated stiffness and pore arrangement variations for guiding cell functions have also been discussed. This review also covers the application of designed patterns, gradients, or topographic modulation on 3D bioprinted structures in fabricating the anisotropic features. Finally, this review accounts for the tissue-specific requirements that can be adopted for topography-motivated enhancement of cellular functions during the fabrication process with a special thrust on bioprinting.

Acute Toxicity Evaluation of the Disinfectant Containing Percarbonate and Tetraacetylethylenediamine by Measuring Behavioral Responses of Small Fish Using Image Analysis.

Disinfectants containing percarbonate and tetraacetylethylenediamine (TAED) has been developed as an effective and relatively safe disinfectant to destroy viruses and bacteria in animals and humans, however it is known that most disinfectants can cause danger to living organisms including humans. In the current study, acute toxicity of the disinfectant composed of percarbonate and TAED was assessed by measuring behavioral responses as well as lethal concentrations of aquatic organisms such as medaka and zebrafish when they were exposed to it. First, the breeding water properties were determined by measuring dissolved oxygen (DO) and pH changes over time up to 96 h in acute toxicity tests using the medaka, and the lethal concentration 50% (LC50, 88.39 ppm) was calculated using the lethality rate of the fish. This experiment was conducted in compliance with traditional OECD guidelines. Second, the assessment of behavioral responses (locomotive activity and swimming speed) with the zebrafish were assessed by the image analysis to capture the images per second for three hours, and the collected data were processed using image analysis to calculate the locomotive activity and swimming speed. Finally, the LC50 (135.76 ppm) of the disinfectant to the fish was also measured after three hours. Overall, the data revealed that LC50 of the disinfectant may be affected by the pH of the water exposed to the disinfectant, not by the DO in the water. In addition, the results from the image analysis indicated that the behavioral responses of the fish can further assess the acute toxicity of the disinfectant at concentrations below the LC50 and there was a relationship (R2 = 0.85) between the behavioral responses and the survival rate of the fish. Electronic Supplementary Material ESM: The online version of this article (doi: 10.1007/s12257-021-0419-0) contains supplementary material, which is available to authorized users.

Effects of Processing Parameters of 3D Bioprinting on the Cellular Activity of Bioinks.

In this review, few established cell printing techniques along with their parameters that affect the cell viability during bioprinting are considered. 3D bioprinting is developed on the principle of additive manufacturing using biomaterial inks and bioinks. Different bioprinting methods impose few challenges on cell printing such as shear stress, mechanical impact, heat, laser radiation, etc., which eventually lead to cell death. These factors also cause alteration of cells phenotype, recoverable or irrecoverable damages to the cells. Such challenges are not addressed in detail in the literature and scientific reports. Hence, this review presents a detailed discussion of several cellular bioprinting methods and their process-related impacts on cell viability, followed by probable mitigation techniques. Most of the printable bioinks encompass cells within hydrogel as scaffold material to avoid the direct exposure of the harsh printing environment on cells. However, the advantages of printing with scaffold-free cellular aggregates over cell-laden hydrogels have emerged very recently. Henceforth, optimal and favorable crosslinking mechanisms providing structural rigidity to the cell-laden printed constructs with ideal cell differentiation and proliferation, are discussed for improved understanding of cell printing methods for the future of organ printing and transplantation.

Systematic approach to characterize the dynamics of protein adsorption on the surface of biomaterials using proteomics.

Protein adsorption on biomaterial surfaces has been investigated in the development of protein-repellent implantable devices. While the study of the adsorption of a single protein have produced valuable insights of the role of specific proteins in the biological responses to biomaterials, a systematic high throughput screening method is needed to gain more comprehensive understanding of such a complex process, mainly because biomaterials are exposed to protein mixtures when implanted in the human body. To further advance our knowledge of the dynamics of protein adsorption/desorption at interfaces between proteins and solid surfaces, proteomic technologies have been explored to determine relationships between adsorbed proteins on the surfaces and subsequent biological responses. In this review, we will briefly describe the protein adsorption process and proteomics technologies and focus on subsequent biological responses to biomaterials such as blood/biomaterial interactions, biocompatibility, and cell behavior, to obtain more comprehensive understanding of the process for the development of improved biomaterials. We also highlight a number of challenges of contemporary proteomics technologies and future perspectives to advance our knowledge of protein adsorption/desorption dynamics on the surfaces of biomaterials.

Evaluation of green tea extract as a safe personal hygiene against viral infections.

BACKGROUND: Viral infections often pose tremendous public health concerns as well as economic burdens. Despite the availability of vaccines or antiviral drugs, personal hygiene is considered as effective means as the first-hand measure against viral infections. The green tea catechins, in particular, epigallocatechin-3-gallate (EGCG), are known to exert potent antiviral activity. In this study, we evaluated the green tea extract as a safe personal hygiene against viral infections. RESULTS: Using the influenza virus A/Puerto Rico/8/34 (H1N1) as a model, we examined the duration of the viral inactivating activity of green tea extract (GTE) under prolonged storage at various temperature conditions. Even after the storage for 56 days at different temperatures, 0.1% GTE completely inactivated 106 PFU of the virus (6 log10 reduction), and 0.01% and 0.05% GTE resulted in 2 log10 reduction of the viral titers. When supplemented with 2% citric acid, 0.1% sodium benzoate, and 0.2% ascorbic acid as anti-oxidant, the inactivating activity of GTE was temporarily compromised during earlier times of storage. However, the antiviral activity of the GTE was steadily recovered up to similar levels with those of the same concentrations of GTE without the supplements, effectively prolonging the duration of the virucidal function over extended period. Cryo-EM and DLS analyses showed a slight increase in the overall size of virus particles by GTE treatment. The results suggest that the virucidal activity of GTE is mediated by oxidative crosslinking of catechins to the viral proteins and the change of physical properties of viral membranes. CONCLUSIONS: The durability of antiviral effects of GTE was examined as solution type and powder types over extended periods at various temperature conditions using human influenza A/H1N1 virus. GTE with supplements demonstrated potent viral inactivating activity, resulting in greater than 4 log10 reduction of viral titers even after storage for up to two months at a wide range of temperatures. These data suggest that GTE-based antiviral agents could be formulated as a safe and environmentally friendly personal hygiene against viral infections.

Mathematical modeling approaches to describe the dynamics of protein adsorption at solid interfaces.

Protein adsorption on solid surfaces play a crucial role in many biological applications. While extensive experimental studies have revealed the dynamics of protein adsorption in detail using newly developed experimental techniques, mathematical modeling approaches provide illustrative insight of such complex processes, which are often difficult to describe experimentally. To advance our understanding of dynamic protein behaviors at interfaces between proteins and solid surfaces, there are needs for additional information about such complex phenomena, especially conformational changes in protein mixture system. In this review, we will briefly describe competitive protein adsorption process (known as 'Vroman effect') and provide a brief overview of some important mathematical modeling approaches to advance our knowledge of the dynamic adsorption/desorption processes and highlight the need for experimental methods to determine the extent of conformational changes and the rate of conformational change of adsorbed proteins at the surfaces.

Study of the conformational change of adsorbed proteins on biomaterial surfaces using hydrogen-deuterium exchange with mass spectroscopy.

There is no doubt that protein adsorption plays a crucial role in determining biocompatibility of biomaterials. Despite the information of the identity and composition of blood plasma/serum proteins adsorbed on surfaces of biomaterials to understand which proteins are involved in blood/biomaterial interactions, it still does not provide information about the conformations and orientations of adsorbed protein, which are very important in determining biological responses to biomaterials. Therefore, our laboratory has developed an experimental technology to probe protein conformations on materials that is applicable to mixtures of proteins. Herein, the new application of hydrogen/deuterium (H/D) exchange combined with mass spectrometry was applied to determine conformational changes of adsorbed proteins at biomaterial surfaces. The results suggest that there may be a significant conformational change in adsorbed proteins at 'low' bulk concentrations that leads to a large change in the kinetics of H/D exchange as compared to 'high' bulk concentrations. This technique may eventually be useful for the study of the kinetics of protein conformational changes.

Tyrosine-derived polycarbonate scaffolds for bone regeneration in a rabbit radius critical-size defect model.

The aim of the study was to determine bone regeneration in a rabbit radius critical-size defect (CSD) model using a specific polymer composition (E1001(1k)) from a library of tyrosine-derived polycarbonate scaffolds coated with a calcium phosphate (CP) formulation (E1001(1k) + CP) supplemented with recombinant human bone morphogenetic protein-2 (rhBMP-2). Specific doses of rhBMP-2 (0, 17, and 35 µg/scaffold) were used. E1001(1k) + CP scaffolds were implanted in unilateral segmental defects (15 mm length) in the radial diaphyses of New Zealand White rabbits. At 4 and 8 weeks post-implantation, bone regeneration was determined using micro-computed tomography (µCT), histology, and histomorphometry. The quantitative outcome data suggest that E1001(1k) + CP scaffolds with rhBMP-2 were biocompatible and promoted bone regeneration in segmental bone defects. Histological examination of the implant sites showed that scaffolds made of E1001(1k) + CP did not elicit adverse cellular or tissue responses throughout test periods up to 8 weeks. Noteworthy is that the incorporation of a very small amount of rhBMP-2 into the scaffolds (as low as 17 µg/defect site) promoted significant bone regeneration compared to scaffolds consisting of E1001(1k) + CP alone. This finding indicates that E1001(1k) + CP may be an effective platform for bone regeneration in a critical size rabbit radius segmental defect model, requiring only a minimal dose of rhBMP-2.

TGF-ß1 conjugated chitosan collagen hydrogels induce chondrogenic differentiation of human synovium-derived stem cells.

BACKGROUND: Unlike bone tissue, articular cartilage regeneration has not been very successful and has many challenges ahead. We have previously developed injectable hydrogels using photopolymerizable chitosan (MeGC) that supported growth of chondrocytes. In this study, we demonstrate a biofunctional hydrogel for specific use in cartilage regeneration by conjugating transforming growth factor-ß1 (TGF-ß1), a well-documented chondrogenic factor, to MeGC hydrogels impregnating type II collagen (Col II), one of the major cartilaginous extracellular matrix (ECM) components. RESULTS: TGF-ß1 was delivered from MeGC hydrogels in a controlled manner with reduced burst release by chemically conjugating the protein to MeGC. The hydrogel system did not compromise viability of encapsulated human synovium-derived mesenchymal stem cells (hSMSCs). Col II impregnation and TGF-ß1 delivery significantly enhanced cellular aggregation and deposition of cartilaginous ECM by the encapsulated cells, compared with pure MeGC hydrogels. CONCLUSIONS: This study demonstrates successful engineering of a biofunctional hydrogel with a specific microenvironment tailored to promote chondrogenesis. This hydrogel system can provide promising efficacious therapeutics in the treatment of cartilage defects.

Next-generation resorbable polymer scaffolds with surface-precipitated calcium phosphate coatings.

Next-generation synthetic bone graft therapies will most likely be composed of resorbable polymers in combination with bioactive components. In this article, we continue our exploration of E1001(1k), a tyrosine-derived polycarbonate, as an orthopedic implant material. Specifically, we use E1001(1k), which is degradable, nontoxic, and osteoconductive, to fabricate porous bone regeneration scaffolds that were enhanced by two different types of calcium phosphate (CP) coatings: in one case, pure dicalcium phosphate dihydrate was precipitated on the scaffold surface and throughout its porous structure (E1001(1k) + CP). In the other case, bone matrix minerals (BMM) such as zinc, manganese and fluoride were co-precipitated within the dicalcium phosphate dihydrate coating (E1001(1k) + BMM). These scaffold compositions were compared against each other and against ChronOS (Synthes USA, West Chester, PA, USA), a clinically used bone graft substitute (BGS), which served as the positive control in our experimental design. This BGS is composed of poly(lactide co-ε-caprolactone) and beta-tricalcium phosphate. We used the established rabbit calvaria critical-sized defect model to determine bone regeneration within the defect for each of the three scaffold compositions. New bone formation was determined after 2, 4, 6, 8 and 12 weeks by micro-computerized tomography (µCT) and histology. The experimental tyrosine-derived polycarbonate, enhanced with dicalcium phosphate dihydrate, E1001(1k) + CP, supported significant bone formation within the defects and was superior to the same scaffold containing a mix of BMM, E1001(1k) + BMM. The comparison with the commercially available BGS was complicated by the large variability in bone formation observed for the laboratory preparations of E1001(1k) scaffolds. At all time points, there was a trend for E1001(1k) + CP to be superior to the commercial BGS. However, only at the 6-week time point did this trend reach statistical significance. Detailed analysis of the µCT data suggested an increase in bone formation from 2 through 12 weeks in implant sites treated with E1001(1k) + CP. At 2 and 4 weeks post-implantation, bone formation occurred at the interface where the E1001(1k) + CP scaffold was in contact with the bone borders of the implant site. Thereafter, during weeks 6, 8 and 12 bone formation progressed throughout the E1001(1k) + CP test implants. This trend was not observed with E1001(1k) + BMM scaffolds or the clinically used BGS. Our results suggest that E1001(1k) + CP should be tested further for osteoregenerative applications.

Visible-light-initiated hydrogels preserving cartilage extracellular signaling for inducing chondrogenesis of mesenchymal stem cells.

Hydrogels have a unique opportunity to regenerate damaged cartilage tissues by introducing mesenchymal stem cells (MSCs) in a highly swollen environment similar to articular cartilage. During cartilage development, collagen-cell interactions play an important role in mediating early mesenchymal condensation and chondrogenesis with transforming growth factor-ß1 (TGF-ß1) stimulation. Here, a hydrogel environment that can enhance cell-matrix interactions and chondrogenesis by stabilizing type-II collagen (Col II) and TGF-ß1 into photopolymerizable (methacrylated) chitosan (MeGC) with simple entrapment and affinity binding is demonstrated. The MeGC hydrogel was designed to gel upon initiation by exposure to visible blue light in the presence of riboflavin, an aqueous initiator from natural vitamin. The incorporation of Col II into MeGC hydrogels increased cellular condensation and deposition of cartilaginous extracellular matrix by encapsulated chondrocytes. MeGC hydrogels containing Col II supported the release of TGF-ß1 in a controlled manner over time in chondrogenic medium and the incorporated TGF-ß1 further enhanced chondrogenesis of encapsulated chondrocytes and MSCs, especially synovial MSCs. Subcutaneous implantation of hydrogel cultures showed greatly improved neocartilage formation in constructs loaded with TGF-ß1 compared with controls. These findings suggest that cartilage mimetic hydrogels have a high potential for cartilage repair.

In vivo performance of combinations of autograft, demineralized bone matrix, and tricalcium phosphate in a rabbit femoral defect model.

Large bone defects may be treated with autologous or allogeneic bone preparations. Each treatment has advantages and disadvantages; therefore, a clinically viable option for treating large (e.g., gap) bone defects may be a combination of the two. In the present study, bone repair was determined with combinations of autografts, allografts, and synthetic bone grafts using an established rabbit femoral defect model. Bilateral unicortical femoral defects were surgically prepared and treated with combinatorial bone grafts according to one of seven treatment groups. Recipient sites were retrieved at six weeks. Cellular/tissue responses and new bone formation were assessed by histology and histomorphometry. Histological analysis images indicated neither evidence of inflammatory, immune responses, tissue necrosis, nor osteolysis. Data suggested co-integration of implanted agents with host and newly formed bone. Finally, the histomorphometric data suggested that the tricalcium phosphate-based synthetic bone graft substitute allowed new bone formation that was similar to the allograft (i.e., demineralized bone matrix, DBM).

Adipose-derived stem cells and BMP-2 delivery in chitosan-based 3D constructs to enhance bone regeneration in a rat mandibular defect model.

Reconstructing segmental mandiblular defects remains a challenge in the clinic. Tissue engineering strategies provide an alternative option to resolve this problem. The objective of the present study was to determine the effects of adipose-derived stem cells (ASCs) and bone morphogenetic proteins-2 (BMP-2) in three-dimensional (3D) scaffolds on mandibular repair in a small animal model. Noggin expression levels in ASCs were downregulated by a lentiviral short hairpin RNA strategy to enhance ASC osteogenesis (ASCs(Nog-)). Chitosan (CH) and chondroitin sulfate (CS), natural polysaccharides, were fabricated into 3D porous scaffolds, which were further modified with apatite coatings for enhanced cellular responses and efficient delivery of BMP-2. The efficacy of 3D apatite-coated CH/CS scaffolds supplemented with ASCs(Nog-) and BMP-2 were evaluated in a rat critical-sized mandibular defect model. After 8 weeks postimplantation, the scaffolds treated with ASCs(Nog-) and BMP-2 significantly promoted rat mandibular regeneration as demonstrated by micro-computerized tomography, histology, and immunohistochemistry, compared with the groups treated with ASCs(Nog-) or BMP-2 alone. These results suggest that our combinatorial strategy of ASCs(Nog-)+BMP-2 in 3D apatite microenvironments can significantly promote mandibular regeneration, and these may provide a potential tissue engineering approach to repair large bony defects.

Preparation of cationic nanogels for nucleic acid delivery.

Cationic nanogels with site-selected functionality were designed for the delivery of nucleic acid payloads targeting numerous therapeutic applications. Functional cationic nanogels containing quaternized 2-(dimethylamino)ethyl methacrylate and a cross-linker with reducible disulfide moieties (qNG) were prepared by activators generated by electron transfer (AGET) atom transfer radical polymerization (ATRP) in an inverse miniemulsion. Polyplex formation between the qNG and nucleic acid exemplified by plasmid DNA (pDNA) and short interfering RNA (siRNA duplexes) were evaluated. The delivery of polyplexes was optimized for the delivery of pDNA and siRNA to the Drosophila Schneider 2 (S2) cell-line. The qNG/nucleic acid (i.e., siRNA and pDNA) polyplexes were found to be highly effective in their capabilities to deliver their respective payloads.

Recombinant human bone morphogenetic protein-2 released from polyurethane-based scaffolds promotes early osteogenic differentiation of human mesenchymal stem cells.

The purposes of this study were to determine the pharmacokinetics of recombinant human bone morphogenetic protein-2 (rhBMP-2) from a polyurethane (PUR)-based porous scaffold and to determine the biological responses of human mesenchymal stem cells (hMSCs) to the rhBMP-2 released from those scaffolds. The rhBMP-2 was incorporated into the PUR three-dimensional (3D) porous scaffolds and release profiles were determined using enzyme-linked immunosorbent assay. The bioactivity of the rhBMP-2 containing releasates was determined using hMSCs and compared with exogenous rhBMP-2. Release of rhBMP-2 from PUR-based systems was bi-phasic and characterized by an initial burst followed by a sustained release for up to 21 days. Expression of alkaline phosphatase activity by hMSCs treated with the rhBMP-2 releasates was significantly greater than the cells alone (control) throughout the time periods. Furthermore, after 14 days of culture, the hMSCs cultured with rhBMP-2 releasate had a greater amount of mineralization compared to exogenous rhBMP-2. Overall, the rhBMP-2 release from the PUR-based scaffolds was sustained for 21 days and the releasates appeared to be bioactive and promoted earlier osteogenic differentiation and mineralization of hMSCs than the exogenous rhBMP-2.

Rapid-prototyped PLGA/ß-TCP/hydroxyapatite nanocomposite scaffolds in a rabbit femoral defect model.

Bone tissue engineering scaffolds composed of poly(d,l-lactide:glycolide) (DL-PLGA) and ß-tricalcium phosphate (ß-TCP) nanocomposites were prepared and characterized. Scaffolds with two specific architectures were produced via fused deposition modeling (FDM), a type of extrusion freeform fabrication. Microfilaments deposited at angles of 0° and 90° were designated as the 'simple' scaffold architecture, while those deposited at angles alternating between 0°, 90°, 45° and -45° were designated as the 'complex' scaffold architecture. In addition, the simple and complex scaffolds were coated with hydroxyapatite (HA). The surface morphology of the scaffolds was assessed before and after HA coating and uniform distribution of HA coating on the surface was observed by scanning electron microscopy. The scaffolds were implanted into rabbit femoral unicortical bone defects according to four treatment groups based on pore structure and HA coating. After 6 and 12 weeks, scaffolds and host bone were recovered and processed for histology. Data suggest that all configurations of the scaffolds integrated with the host bone and were biocompatible and thus may offer an exciting new scaffold platform for delivery of biologicals for bone regeneration.

Bone regeneration in a rabbit critical-sized calvarial model using tyrosine-derived polycarbonate scaffolds.

Porous three-dimensional tyrosine-derived polycarbonate (TyrPC) scaffolds with a bimodal pore distribution were fabricated to mimic bone architecture using a combination of salt-leaching and phase separation techniques. TyrPC scaffolds degraded in register with bone regeneration during the 6-week study period and compressive moduli of the scaffolds were maintained >0.5 MPa at 6 weeks of incubation in PBS at 37 °C. The TyrPC scaffolds either unsupplemented or supplemented with recombinant human bone morphogenetic protein-2 (rhBMP-2) were implanted in a rabbit calvarial critical-sized defect (CSD) model and the TyrPC scaffolds treated with rhBMP-2 or TyrPC coated with calcium phosphate scaffold alone promoted bone regeneration in a rabbit calvarial CSD at 6 weeks postimplantation. A synthetic TyrPC polymeric scaffold either without a biological supplement or with a minimal dose of rhBMP-2 induced bone regeneration comparable to a commercially available bone graft substitute in a nonrodent CSD animal model.

Fiber-reinforced calcium phosphate cement formulations for cranioplasty applications: a 52-week duration preclinical rabbit calvaria study.

The in vivo tissue response to a newly developed fiber-reinforced calcium phosphate cement (CPC) formulation was assessed using a well-established rabbit calvarial defect model. Bilateral subcritical sized (8-mm diameter) defects were surgically created in the parietal bones of each rabbit (a total of 48 rabbits), and randomized to be filled with either the new fiber-reinforced formulation, a conventional CPC (positive control), or left unfilled (negative control). The implant sites were subsequently retrieved after 12, 24, and 52 weeks postsurgery. Each specimen, including the parietal bone craniotomy and underlying brain, were recovered at necropsy and the tissue responses were assessed by histology. The resulting histological slides indicated that there was no evidence of severe inflammatory responses or osteolysis. The data showed new dural and pericranial bone formation along the implants, as well as excellent bone-to-implant interfaces in all of the CPC-filled defects. These results suggest that the biologic response to the new fiber-reinforced CPC formulations and conventional nonreinforced CPC are very similar, and both demonstrate excellent biocompatibility as well as an overall osteophylic response.

Osteoblast growth and bone-healing response to three-dimensional poly(ε-caprolactone fumarate) scaffolds.

Poly(ε-caprolactone fumarate) (PCLF) scaffold formulations were assessed as a delivery system for recombinant human bone morphogenetic protein (rhBMP-2) for bone tissue engineering. The formulations included PCLF with combinations of poly(vinyl alcohol) (PVA) and hydroxyapatite (HA). The assessments included in vitro and in vivo assays. In vitro assays validated cell attachment using a pre-osteoblast cell line (MC3T3-E1). Additionally, in vitro release profiles of rhBMP-2 from PCLF scaffolds were determined up to 21 days. The data suggested that PCLF incorporated with PVA and HA accelerated rhBMP-2 release and that the released protein was bioactive. For the in vivo study, a critical-sized defect (CSD) model in rabbit calvaria was used to test PCLF scaffolds. At 6 weeks post-implantation, significantly more bone formation was measured in PCLF scaffolds containing rhBMP-2 than in scaffolds without rhBMP-2. In conclusion, we demonstrated that PCLF delivered biologically active rhBMP-2, promoted bone healing in a CSD and has potential as a bone tissue engineering scaffold.