Improved biological performance of human cartilage-derived progenitors in platelet lysate xenofree media in comparison to fetal bovine serum media.

Primary articular cartilage-derived cells are among the preferred contenders for cell-based therapy approaches for cartilage repair. Limited access to primary human cartilage tissue necessitates the process of in vitro cell expansion to obtain sufficient cells for therapeutic purposes. Therapeutic outcomes of such cell-based approaches become highly dependent on the quality of the in vitro culture-expanded cells. The objective of this study was to determine the differential biological effects of human platelet lysate (hPL) xeno-free defined media vs FBS containing traditional media on primary human cartilage-derived cells. Our goal in pursuing this work was to identify a preferred xenofree media alternative, that can be used as a platform for expansion of cells intended for clinical applications. Primary cartilage-derived cells obtained from five patients were simultaneously cultured in two expansion media's: (1) traditional (DMEM+10%FBS+1%P/S) and (2) defined xenofree (Nutristem® complete media+0.5%hPL). Connective tissue progenitors (CTPs) were assayed by standard colony forming unit assay, morphology, proliferation in early and late passages, expression of MSC associated cell-surface markers (CD73, CD90 and CD105) and trilineage differentiation (adipogenesis, osteogenesis and chondrogenesis) were considered for comparison of biological performance. Early biological performance of primary cartilage-derived cells was significantly improved in Nutristem® expansion media in comparison to traditional expansion media with respect to (1) Colony forming efficiency tended to be higher (p = 0.058) and (2) CTPs formed larger colonies with respect to total cells per colony and colony area (p < 0.01). In the culture expanded cell population, Nutristem® expansion media was superior to traditional expansion media with respect to: (1) overall proliferation rate through passages 1-4 (p = 0.027), (2) total cells harvested at end of passage 4 (p = 0.028) and (3) total positive stain area of CD73 (p = 0.006), CD90 (p = 0.001) and CD105 (p = 0.049). Nutristem®-hPL expanded cells when differentiated in respective xenofree serum-free defined MSCgo™ differentiated media's, also showed significant improvement in adipogenic, osteogenic and chondrogenic marker expression. Overall, we convincingly demonstrated that a low concentration of hPL in combination with defined xenofree media is an effective and economic growth supplement to culture expand primary cartilage-derived cells. It can be manufactured under cGMP conditions to improve clinical-grade cell products' quality for therapeutic applications.

Assessment of Clinical, Tissue, and Cell-Level Metrics Identify Four Biologically Distinct Knee Osteoarthritis Patient Phenotypes.

OBJECTIVE: Clinical heterogeneity of primary osteoarthritis (OA) is a major challenge in understanding pathogenesis and development of targeted therapeutic strategies. This study aims to (1) identify OA patient subgroups phenotypes and (2) determine predictors of OA severity and cartilage-derived stem/progenitor concentration using clinical-, tissue-, and cell- level metrics. DESIGN: Cartilage, synovium (SYN) and infrapatellar fatpad (IPFP) were collected from 90 total knee arthroplasty patients. Clinical metrics (patient demographics, radiograph-based joint space width (JSW), Kellgren and Lawrence score (KL)), tissue metrics (cartilage histopathology grade, glycosaminoglycans (GAGs)) and cell-based metrics (cartilage-, SYN-, and IPFP-derived cell concentration ([Cell], cells/mg), connective tissue progenitor (CTP) prevalence (PCTP, CTPs/million cells plated), CTP concentration, [CTP], CTPs/mg)) were assessed using k-mean clustering and linear regression model. RESULTS: Four patient subgroups were identified. Clusters 1 and 2 comprised of younger, high body mass index (BMI) patients with healthier cartilage, where Cluster 1 had high CTP in cartilage, SYN, and IPFP, and Cluster 2 had low [CTP] in cartilage, SYN, and IPFP. Clusters 3 and 4 comprised of older, low BMI patients with diseased cartilage where Cluster 3 had low [CTP] in SYN, IPFP but high [CTP] in cartilage, and Cluster 4 had high [CTP] in SYN, IPFP but low [CTP] in cartilage. Age (r = 0.23, P = 0.026), JSW (r = 0.28, P = 0.007), KL (r = 0.26, P = 0.012), GAG/mg cartilage tissue (r = -0.31, P = 0.007), and SYN-derived [Cell] (r = 0.25, P = 0.049) were weak but significant predictors of OA severity. Cartilage-derived [Cell] (r = 0.38, P < 0.001) and PCTP (r = 0.9, P < 0.001) were moderate/strong predictors of cartilage-derived [CTP]. CONCLUSION: Initial findings suggests the presence of OA patient subgroups that could define opportunities for more targeted patient-specific approaches to prevention and treatment.

A comprehensive dataset of histopathology images, grades and patient demographics for human Osteoarthritis Cartilage.

Osteoarthritis (OA) is a leading cause of disability in older adults and takes substantial toll at personal, economic and societal levels. There is inadequate comprehension of OA disease progression specifically during the early phases of OA. This knowledge is critical to understanding the heterogeneity in OA progression as well as enable development of targeted therapeutics at the start of the disease rather than end-stage. Histopathology of cartilage is a common method used to assess in situ state of cartilage tissue. The data presented in this article assesses the histopathological status of human cartilage specimens collected from 90 patients (n = 180). Each specimen was processed for histology and stained with hematoxylin and eosin (HE) and safranin O fast-green (SafO) for acquiring brightfield images to visualize changes in cartilage structure, cells, gycosaminoglycan content and tidemark integrity. The unstained sections were imaged using polarized light microscopy (PLM) to visualize changes in collagen organization and composition within the cartilage specimen. All the specimens were systematically graded by three scorers using established primary OA cartilage grading systems including Histological-Histochemical Grading System (HHGS), advanced Osteoarthritis Research Society International (OARSI) system and Polarized Light Microscopy (PLM) scoring system. These data can be used by the OA community as an educational resource to train new reviewers (scorers), it serves as a comprehensive image database for experienced OA community to review the wide spectrum of histopathological features presented by these mild to moderate OA specimens, to define different OA-subtypes, and to generate hypothesis on OA progression mechanisms. Finally, the high quality images can be used to develop machine learning algorithms for classification of OA, automated detection and segmentation of existing or new OA features that can serve as early OA histopathological indicators.

Patient Age and Cell Concentration Influence Prevalence and Concentration of Progenitors in Bone Marrow Aspirates: An Analysis of 436 Patients.

BACKGROUND: Connective tissue progenitors (CTPs) resident in native tissues serve as biological building blocks in tissue repair and remodeling processes. Methods for analysis and reporting on CTP quantity and quality are essential for defining optimal cell sources and donor characteristics and the impact of cell processing methods for cell therapy applications. The present study examines the influence of donor characteristics and cell concentration (nucleated cells/mL) on CTP prevalence (CTPs/million nucleated cells) and CTP concentration (CTPs/mL) in bone marrow aspirates (BMAs). METHODS: Iliac crest bone marrow was aspirated from 436 patients during elective total knee or hip arthroplasty. Bone marrow-derived nucleated cells were plated at a density of 1.19 × 105 cells/cm2. Colony-forming unit analysis was performed on day 6. RESULTS: Large variation was seen between donors. Age (p < 0.05) and cell concentration (p < 0.001) significantly influenced CTP prevalence and CTP concentration. For every 1-year increase in age, the odds of having at least an average CTP prevalence and CTP concentration decreased by 1.5% and 1.6%, respectively. For every 1 million cells/mL increase in cell concentration, the odds of having at least an average CTP prevalence and CTP concentration increased by 2.2% and 7.9%, respectively. Sex, race, body mass index (BMI), and the presence of osteoporosis did not influence CTP prevalence or CTP concentration. CONCLUSIONS: BMA-derived CTPs were obtained from all patient groups. CTP prevalence and CTP concentration decreased with age. Cell concentration decreased with age and positively correlated with total CTP prevalence and CTP concentration. The mean CTP concentration in patients >60 years of age was a third of the CTP concentration in patients <30 years of age. CLINICAL RELEVANCE: Proper BMA techniques are necessary to obtain a high-quality yield and composition of cells and CTPs. The reduced CTP concentration and CTP prevalence in the elderly may be mitigated by the use of cell processing methods that increase CTP concentration and CTP prevalence (e.g., by removing red blood cells, serum, and non-CTPs or by increasing aspirate volumes). Cell concentration in the BMA can be measured at the point of care and is an appropriate initial assessment of the quality of BMA.

Characterization of heterogeneous primary human cartilage-derived cell population using non-invasive live-cell phase-contrast time-lapse imaging.

Reliable and reproducible cell therapy strategies to treat osteoarthritis demand an improved characterization of the cell and heterogeneous cell population resident in native cartilage tissue. Using live-cell phase-contrast time-lapse imaging (PC-TLI), this study investigates the morphological attributes and biological performance of the three primary biological objects enzymatically isolated from primary human cartilage: connective tissue progenitors (CTPs), non-progenitors (NPs) and multi-cellular structures (MCSs). The authors' results demonstrated that CTPs were smaller in size in comparison to NPs (P < 0.001). NPs remained part of the adhered cell population throughout the cell culture period. Both NPs and CTP progeny on day 8 increased in size and decreased in circularity in comparison to their counterparts on day 1, although the percent change was considerably less in CTP progeny (P < 0.001). PC-TLI analyses indicated three colony types: single-CTP-derived (29%), multiple-CTP-derived (26%) and MCS-derived (45%), with large heterogeneity with respect to cell morphology, proliferation rate and cell density. On average, clonal (CL) (P = 0.009) and MCS (P = 0.001) colonies exhibited higher cell density (cells per colony area) than multi-clonal (MC) colonies; however, it is interesting to note that the behavior of CL (less cells per colony and less colony area) and MCS (high cells per colony and high colony area) colonies was quite different. Overall effective proliferation rate (EPR) of the CTPs that formed CL colonies was higher than the EPR of CTPs that formed MC colonies (P = 0.02), most likely due to CTPs with varying EPR that formed the MC colonies. Finally, the authors demonstrated that lag time before first cell division of a CTP (early attribute) could potentially help predict its proliferation rate long-term. Quantitative morphological characterization using non-invasive PC-TLI serves as a reliable and reproducible technique to understand cell heterogeneity. Size and circularity parameters can be used to distinguish CTP from NP populations. Morphological cell and colony features can also be used to reliably and reproducibly identify CTP subpopulations with preferred proliferation and differentiation potentials in an effort to improve cell manufacturing and therapeutic outcomes.

Influence of Glucose Concentration on Colony-Forming Efficiency and Biological Performance of Primary Human Tissue-Derived Progenitor Cells.

OBJECTIVE: Glucose concentrations used in current cell culture methods are a significant departure from physiological glucose levels. The study focuses on comparing the effects of glucose concentrations on primary human progenitors (connective tissue progenitors [CTPs]) used for cartilage repair. DESIGN: Cartilage- (Outerbridge grade 1, 2, 3; superficial and deep zone cartilage), infrapatellar fatpad-, synovium-, and periosteum-derived cells were obtained from 63 patients undergoing total knee arthroplasty and cultured simultaneously in fresh chondrogenic media containing 25 mM glucose (HGL) or 5 mM glucose (NGL) for pairwise comparison. Automated ASTM-based quantitative image analysis was used to determine colony-forming efficiency (CFE), effective proliferation rates (EPR), and sulfated-proteoglycan (GAG-ECM) staining of the CTPs across tissue sources. RESULTS: HGL resulted in increased cell cultures with CFE = 0 compared with NGL in all tissue sources (P = 0.049). The CFE in NGL was higher than HGL for superficial cartilage (P < 0.001), and contrary for synovium-derived CTPs (P = 0.046) when CFE > 0. EPR of the CTPs did not differ between the media in the 6-day assay time period (P = 0.082). The GAG-ECM area of the CTPs and their progeny was increased in presence of HGL (P = 0.027). CONCLUSION: Glucose concentration is critical to progenitor's physiology and should be taken into account in the setting of protocols for clinical or in vitro cell expansion strategies.

Automated in-process characterization and selection of cell-clones for quality and efficient cell manufacturing.

Delivery of safe, effective and reliable cellular therapies, whether based on mesenchymal stromal cells (MSCs) or induced pluripotent stem cells (iPSCs), demand standardization of cell culture protocols. There is a need to develop automation platform that enables the users to generate culture expanded human cell populations that improves the quality and reduces batch-to-batch variation with respect to biological potential. Cell X™ robot was designed to address these current challenges in the cell fabrication industry. It utilizes non-invasive large field of view quantitative image analysis to guide an automated process of targeted "biopsy" (cells or media), "picking" (selection) of desired cells or colonies, or "weeding" (removal) of undesired cells, thus providing an unprecedented ability to acquire quantitative measurement in a complex heterogeneous cell environment "in process" and then to act on those measurements to define highly reproducible methods for cell and colony "management" based on application specific critical quality attributes to improve the quality of the manufactured cell lines and cell products.

Native-Osteoarthritic Joint Resident Stem and Progenitor Cells for Cartilage Cell-Based Therapies: A Quantitative Comparison With Respect to Concentration and Biological Performance.

BACKGROUND: Cell-based therapy for cartilage repair is a promising approach and is becoming an established technique. Yet, there is no consensus on the optimal cell source. PURPOSE: To provide a donor-matched quantitative comparison of the connective tissue progenitors (CTPs) derived from cartilage (Outerbridge grade 1-3 [G1-2-3]), bone marrow aspirate concentrate (BMC), infrapatellar fat pad (IPFP), synovium, and periosteum with respect to (1) cell concentration ([Cell], cells/mL), (2) CTP prevalence (PCTP, colonies per million cells), and (3) biological performance based on in vitro proliferation potential (cells per colony) colony density, and differentiation potential (expression of negatively charged extracellular matrix: glycosaminoglycan-rich extra cellular matrix [GAG-ECM]). STUDY DESIGN: Descriptive laboratory study. METHODS: Tissues were obtained from 10 patients undergoing total knee arthroplasty (mean age, 59 years; women, n = 6). Automated quantitative colony-forming unit analysis was used to compare [Cell], PCTP, and CTP biological performance across tissue sources. RESULTS: [Cell] was highest in grade 3 cartilage (P = .002) and BMC (P = .001). Median PCTP was highest in IPFP (P = .001), synovium (P = .003), and G1-2 cartilage (P = .02). Proliferation was highest in synovium-derived CTPs (P < .001). Median colony density was highest in G1-2-3 (P < .001). Median GAG-ECM was highest in G1-2-3 (P < .001). Within each patient, CTPs derived from all tissues were highly heterogeneous in biological performance as determined by cells per colony, density, and GAG-ECM. CONCLUSION: Tissue sources differ in [Cell], PCTP, and biological attributes. The data presented in this study suggest that cartilage (G1-2-3) is the preferred tissue source for cartilage repair based on PCTP and GAG-ECM, followed by synovium, IPFP, BMC, and periosteum. However, due to the heterogeneous mixture of CTPs within each tissue source, there exists a subset of CTPs with biological performance similar to G1-2-3 cartilage, particularly in synovium and IPFP. Performance-based clonal selection and expansion of preferred CTPs and their progeny will potentially lead to improved cell population with predictive future. CLINICAL RELEVANCE: Optimal tissue regeneration strategies will require informed decisions regarding which of the available tissue sources to use. Optimizing cell sourcing in any tissue may require separation of CTPs with preferred attributes from those with less desirable attributes. The heterogeneity manifest in the early stage of colony formation represents an opportunity for performance-based clone selection for clinical cell processing and manufacturing.

Reliable assessment of bone marrow and bone marrow concentrates using automated hematology analyzer.

Aim: A limiting factor in advancement of bone marrow based cell therapies is the lack of characterization of cell products delivered to patients. Methods: Using an automated hematology analyzer that can be implemented in clinical setting, the composition of bone marrow aspirates (n = 17 patients) and bone marrow concentrates (n = 12 patients) were assessed. ICC estimates were calculated for measuring reliability. Results: Bone marrow aspirates assessment resulted in excellent reliability for determining white blood cells (ICC - 0.96; 95% CI: 0.92-0.99), red blood cells (ICC - 0.9; 95% CI: 0.77-0.96), platelets (ICC - 0.93; 95% CI: 0.85-0.97) composition. Bone marrow concentrate assessment resulted in excellent reliability for determining white blood cells (ICC - 0.97; 95% CI: 0.93-0.99), platelets (ICC - 0.95; 95% CI: 0.89-0.99) and moderate reliability for red blood cells (ICC - 0.66; 95% CI: 0.36-0.87) composition. Conclusion: Modern automated hematology analyzers could assist to better characterize the cell therapy products to provide reliable and consistent outcomes.

Variability in the Preparation, Reporting, and Use of Bone Marrow Aspirate Concentrate in Musculoskeletal Disorders: A Systematic Review of the Clinical Orthopaedic Literature.

BACKGROUND: Interest in the therapeutic potential of bone marrow aspirate concentrate (BMAC) has grown exponentially. However, comparisons among studies and their processing methods are challenging because of inconsistent reporting of protocols, as well as poor characterization of the composition of the initial bone marrow aspirate and of the final products delivered. The purpose of this study was to perform a systematic review of the literature to evaluate the level of reporting related to the protocols used for BMAC preparation and the composition of BMAC utilized in the treatment of musculoskeletal diseases in published clinical studies. METHODS: A systematic review of the literature was performed by searching PubMed, MEDLINE, the Cochrane Database of Systematic Reviews, and the Cochrane Central Register of Controlled Trials from 1980 to 2016. Inclusion criteria were human clinical trials, English language, and manuscripts that reported on the use of BMAC in musculoskeletal conditions. RESULTS: After a comprehensive review of the 986 identified articles, 46 articles met the inclusion criteria for analysis. No study provided comprehensive reporting that included a clear description of the preparation protocol that could be used by subsequent investigators to repeat the method. Only 14 (30%) of the studies provided quantitative metrics of the composition of the BMAC final product. CONCLUSIONS: The reporting of BMAC preparation protocols in clinical studies was highly inconsistent and studies did not provide sufficient information to allow the protocol to be reproduced. Moreover, comparison of the efficacy and yield of BMAC products is precluded by deficiencies in the reporting of preparation methods and composition. Future studies should contain standardized and stepwise descriptions of the BMAC preparation protocol, and the composition of the BMAC delivered, to permit validating and rationally optimizing the role of BMAC in musculoskeletal care.

High occurrence of osteoarthritic histopathological features unaccounted for by traditional scoring systems in lateral femoral condyles from total knee arthroplasty patients with varus alignment.

Background and purpose - A better understanding of the patterns and variation in initiation and progression of osteoarthritis (OA) in the knee may influence the design of therapies to prevent or slow disease progression. By studying cartilage from the human lateral femoral condyle (LFC), we aimed to: (1) assess specimen distribution into early, mild, moderate, and severe OA as per the established histopathological scoring systems (HHGS and OARSI); and (2) evaluate whether these 2 scoring systems provide sufficient tools for characterizing all the features and variation in patterns of OA. Patients and methods - 2 LFC osteochondral specimens (4 x 4 x 8 mm) were collected from 50 patients with idiopathic OA varus knee and radiographically preserved lateral compartment joint space undergoing total knee arthroplasty. These were fixed, sectioned, and stained with HE and Safranin O-Fast Green (SafO). Results - The histopathological OA severity distribution of the 100 specimens was: 6 early, 62 mild, 30 moderate, and 2 severe. Overall, 45/100 specimens were successfully scored by both HHGS and OARSI: 12 displayed low OA score and 33 displayed cartilage surface changes associated with other histopathological features. However, 55/100 samples exhibited low surface structure scores, but were deemed to be inadequately scored by HHGS and OARSI because of anomalous features in the deeper zones not accounted for by these systems: 27 exhibited both SafO and tidemark abnormal features, 16 exhibited only SafO abnormal features, and 12 exhibited tidemark abnormal features. Interpretation - LFC specimens were scored as mild to moderate OA by HHGS and OARSI. Yet, several specimens exhibited deep zone anomalies while maintaining good surface structure, inconsistent with mild OA. Overall, a better classification of these anomalous histopathological features could help better understand idiopathic OA and potentially recognize different subgroups of disease.

Progenitor cells from different zones of human cartilage and their correlation with histopathological osteoarthritis progression.

Cell-based therapies development for the treatment of osteoarthritis (OA) requires an understanding of the disease progression and attributes of the cells resident in cartilage. This study focused on quantitative assessment of the concentration and biological potential of stem and progenitor cells resident in different zones of cartilage displaying macroscopic Outerbridge grade 1-2 OA, and their correlation with OA progression based on established histologic scoring system. Lateral femoral condyles were collected from 15 patients with idiopathic OA and varus knees undergoing total knee arthroplasty. Superficial(Csp , top ∼ 500 µm) and deep cartilage(Cdp ) was separated. Chondrogenic Connective Tissue Progenitors (CTP-C) were assayed by standardized Colony-Forming-Unit assay using automated image analysis (ColonyzeTM ) based on ASTM standard F-2944-12. Cell concentration (cells/mg) was significantly greater in Csp (median: 7,000; range: 3,440-17,600) than Cdp (median: 5,340; range: 3,393-9,660), p = 0.039. Prevalence (CTPs/million cells) was not different between Csp (median: 1,274; range: 0-3,898) and Cdp (median:1,365; range:0-6,330), p = 0.42. In vitro performance of CTP-C progeny varied widely within and between patients, manifest by variation in colony size and morphology. Mean histopathological Mankin score was 4.7 (SD = 1.2), representing mild to moderate OA. Tidemark breach by blood vessels was associated with lower Csp cell concentration (p = 0.02). Matrix degradation was associated with lower Cdp cell and CTP-C concentration (p = 0.015 and p = 0.095, respectively), independent of articular surface changes. These findings suggest that the initiation of OA may occur in either superficial or deep zones. The pathological changes affect CTP-Cs in Csp and Cdp cartilage zones differently. The heterogeneity among the available CTP-Cs in Csp and Cdp suggests performance-based selection to optimize cell-sourcing strategies for therapy. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:1728-1738, 2018.

Cellular Therapies in Orthopedics: Where Are We?

The orthopedic field has experienced several major practice-changing pivotal shifts in the past several decades, such as the invention and application of the arthroscope or the implementation and advancement of joint arthroplasties. Most of these previous breakthroughs have focused on surgical techniques and devices. However, the next major advance in the field is likely to be related to biologic treatments. Although still in its early stage of development, orthopedic regenerative medicine, including cellular therapies, represents a great opportunity, since we are only beginning to understand their biological potential. The main challenge in this pathway is to translate the promising results obtained by basic scientists to clinical practice. This work reviewed the market and clinical evidence, as well as future perspectives, concerning cellular therapies in orthopedics.

Effect of dual delivery of antibiotics (vancomycin and cefazolin) and BMP-7 from chitosan microparticles on Staphylococcus epidermidis and pre-osteoblasts in vitro.

The main aims of this manuscript are to: i) determine the effect of commonly used antibiotics to treat osteoarticular infections on osteoblast viability, ii) study the dual release of the growth factor (BMP-7) and antibiotics (vancomycin and cefazolin) from chitosan microparticles iii) demonstrate the bioactivity of the antibiotics released in vitro on Staphylococcus epidermidis. The novelty of this work is dual delivery of growth factor and antibiotic from the chitosan microparticles in a controlled manner without affecting their bioactivity. Cefazolin and vancomycin have different therapeutic concentrations for their action in vivo and therefore, two different concentrations of the drugs were used. Osteoblast cytotoxicity test concluded that cefazolin concentrations of 50 and 100µg/ml were found to have positive influence on osteoblast proliferation. A significant increase in osteoblast proliferation was observed in the presence of cefazolin and BMP-7 in comparison with BMP-7 alone group; indicating cefazolin might play a role in osteoblast proliferation. On the other hand, vancomycin concentration of 1000µg/ml was found to significantly reduce (p<0.01) osteoblast proliferation in comparison with controls. The microbial study indicated that cefazolin at a minimum concentration of 21.5µg/ml could inhibit ~85% growth of S. epidermidis, whereas vancomycin at a concentration of 30µg/ml was found to inhibit ~80% bacterial growth.

Bone regeneration using injectable BMP-7 loaded chitosan microparticles in rat femoral defect.

Injectable chitosan microparticles were prepared using a simple coacervation method under physiologically friendly conditions by eliminating oil or toxic chemical, and employing low temperature and pressure for growth factor stability. Amount of 200 ng of bone morphogenetic protein-7 (BMP-7) was incorporated in the chitosan microparticles by two methods: encapsulating and coating techniques. These microparticles were tested in vivo to determine the biological response in a rat femoral bone defect at 6 and 12 weeks. Four groups (n=10) were tested which include two groups for BMP-7 incorporated microparticles (by two techniques), microparticles without BMP-7, and defect itself (negative control). Healthy bone formation was observed around the microparticles, which were only confined to the defect site and did not disperse. Histology indicated minor inflammatory response around the microparticles at 6 weeks, which reduced by 12 weeks. Micro-CT analysis of bone surface density and porosity was found to be significantly more (p<0.05) for microparticles containing groups, in comparison with controls, which suggests that the new bone formed in the presence of microparticles is more interconnected and porous. Collagen fibrils analysis conducted using multiphoton microscopy showed significant improvement in the formation of bundled collagen area (%) in microparticles containing groups in comparison with controls, indicating higher cross-linking between the fibrils. Microparticles were biocompatible and did not degrade in the 12 week implant period.

IGF-1 release kinetics from chitosan microparticles fabricated using environmentally benign conditions.

The main objective of this study is to maximize growth factor encapsulation efficiency into microparticles. The novelty of this study is to maximize the encapsulated growth factors into microparticles by minimizing the use of organic solvents and using relatively low temperatures. The microparticles were fabricated using chitosan biopolymer as a base polymer and cross-linked with tripolyphosphate (TPP). Insulin like-growth factor-1 (IGF-1) was encapsulated into microparticles to study release kinetics and bioactivity. In order to authenticate the harms of using organic solvents like hexane and acetone during microparticle preparation, IGF-1 encapsulated microparticles prepared by the emulsification and coacervation methods were compared. The microparticles fabricated by emulsification method have shown a significant decrease (p<0.05) in IGF-1 encapsulation efficiency, and cumulative release during the two-week period. The biocompatibility of chitosan microparticles and the bioactivity of the released IGF-1 were determined in vitro by live/dead viability assay. The mineralization data observed with von Kossa assay, was supported by mRNA expression levels of osterix and runx2, which are transcription factors necessary for osteoblasts differentiation. Real time RT-PCR data showed an increased expression of runx2 and a decreased expression of osterix over time, indicating differentiating osteoblasts. Chitosan microparticles prepared in optimum environmental conditions are a promising controlled delivery system for cells to attach, proliferate, differentiate and mineralize, thereby acting as a suitable bone repairing material.

Injectable chitosan microparticles incorporating bone morphogenetic protein-7 for bone tissue regeneration.

This study investigates the influence of the controlled release of bone morphogenetic protein 7 (BMP-7) from cross-linked chitosan microparticles on pre-osteoblasts (OB-6) in vitro. BMP-7 was incorporated into microparticles by encapsulation during the particle preparation and coating after particle preparation. Chitosan microparticles had an average diameter of 700 µm containing ∼10-15 ng of BMP-7. The release study profile indicates that nearly 98% of the BMP-7 coated on the microparticles was released in a period of 18 days while only 36% of the BMP-7 encapsulated in the microparticles was released in the same time period. Cell attachment study indicated that the BMP-7 coated microparticles have many cells adhered on the microparticles in comparison with microparticles without growth factors on day 10. DNA assay indicated a statistical significant increase (p < 0.05) in the amount of DNA obtained from BMP-7 encapsulated and coated microparticles in comparison with microparticles without any growth factors. A real-time RT-PCR experiment was performed to determine the expression of a few osteoblast specific genes-Dlx5, runx2, osterix, osteopontin, osteocalcin, and bone sialoprotein. The results thus suggest that chitosan microparticles obtained by coacervation method are biocompatible and helps in improving the encapsulation efficiency of BMP-7. Also BMP-7 incorporated in the microparticles is being released in a controlled fashion to support attachment, proliferation and differentiation of pre-osteoblasts, thus acting as a good scaffold for bone tissue regeneration.

An overview of recent advances in designing orthopedic and craniofacial implants.

Great deal of research is still going on in the field of orthopedic and craniofacial implant development to resolve various issues being faced by the industry today. Despite several disadvantages of the metallic implants, they continue to be used, primarily because of their superior mechanical properties. In order to minimize the harmful effects of the metallic implants and its by-products, several modifications are being made to these materials, for instance nickel-free stainless steel, cobalt-chromium and titanium alloys are being introduced to eliminate the toxic effects of nickel being released from the alloys, introduce metallic implants with lower modulus, reduce the cost of these alloys by replacing rare elements with less expensive elements etc. New alloys like tantalum, niobium, zirconium, and magnesium are receiving attention given their satisfying mechanical and biological properties. Non-oxide ceramics like silicon nitride and silicon carbide are being currently developed as a promising implant material possessing a combination of properties such as good wear and corrosion resistance, increased ductility, good fracture and creep resistance, and relatively high hardness in comparison to alumina. Polymer/magnesium composites are being developed to improve mechanical properties as well as retain polymer's property of degradation. Recent advances in orthobiologics are proving interesting as well. This paper thus deals with the latest improvements being made to the existing implant materials and includes new materials being introduced in the field of biomaterials.