Effect of local anesthetics on viability and differentiation of various adult stem/progenitor cells

Background Local anesthetics (LAs) are widely used to control pain during various clinical treatments. One of the side effects of LAs, cytotoxicity, has been investigated in various cells including stem/progenitor cells. However, our understanding of the effects of LAs on the differentiation capacity of stem/progenitor cells still remains limited. Therefore, a comparative study was conducted to investigate the effects of multiple LAs on viability and multi-lineage differentiation of stem/progenitor cells that originated from various adult tissues. Method Multiple types of stem/progenitor cells, including bone marrow mesenchymal stem/progenitor cells (MSCs), dental pulp stem/progenitor cells (DPSCs), periodontal ligament stem/progenitor cells (PDLSCs), and tendon-derived stem/progenitor cells, were either obtained from a commercial provider or isolated from adult human donors. Lidocaine (LD) and bupivacaine (BP) at various doses (1×, 0.75×, 0.5×, and 0.25× of each physiological dose) were applied to the different stem/progenitor cells for an hour, followed by induction of fibrogenic, chondrogenic, osteogenic, and adipogenic differentiation. Live/dead and MTT assays were performed at 24 h after the LD or BP treatment. At 2 weeks, qRT-PCR was conducted to evaluate the gene expressions associated with differentiation. After 4 weeks, multiple biochemical staining was performed to evaluate matrix deposition. Results At 24 h after LD or BP treatment, 1× and 0.75× physiological doses of LD and BP showed significant cytotoxicity in all the tested adult stem/progenitor cells. At 0.5×, BP resulted in higher viability than the same dose LD, with variance between cell types. Overall, the gene expressions associated with fibrogenic, chondrogenic, osteogenic, and adipogenic differentiation were attenuated in LD or BP pre-treated stem/progenitor cells, with notable dose-effect and dependence on types. In contrast, certain doses of LD and/or BP were found to increase specific gene expression, depending on the cell types. Conclusion Our data suggest that LAs such as LD and BP affect not only the viability but also the differentiation capacity of adult stem/progenitor cells from various anatomical sites. This study sheds light on stem cell applications for tissue regeneration in which isolation and transplantation of stem cells frequently involve LA administration

Engineered Healing of Avascular Meniscus Tears by Stem Cell Recruitment

Meniscus injuries are extremely common with approximately one million patients undergoing surgical treatment annually in the U.S. alone. Upon injury, the outer zone of the meniscus can be repaired and expected to functionally heal but tears in the inner avascular region are unlikely to heal. To date, no regenerative therapy has been proven successful for consistently promoting healing in inner-zone meniscus tears. Here, we show that controlled applications of connective tissue growth factor (CTGF) and transforming growth factor beta 3 (TGFβ3) can induce seamless healing of avascular meniscus tears by inducing recruitment and step-wise differentiation of synovial mesenchymal stem/progenitor cells (syMSCs). A short-term release of CTGF, a selected chemotactic and profibrogenic cue, successfully recruited syMSCs into the incision site and formed an integrated fibrous matrix. Sustain-released TGFβ3 then led to a remodeling of the intermediate fibrous matrix into fibrocartilaginous matrix, fully integrating incised meniscal tissues with improved functional properties. Our data may represent a novel clinically relevant strategy to improve healing of avascular meniscus tears by recruiting endogenous stem/progenitor cells

Physicomechanical, In Vitro and In Vivo Performance of 3D Printed Doped Tricalcium Phosphate Scaffolds for Bone Tissue Engineering and Drug Delivery

Although tricalcium phosphate (TCP) is widely used in bone tissue engineering, the strength degradation kinetics is not well controlled. This study focuses on the underlying mechanism of strength degradation kinetics by incorporating trace elements in TCP. The objective of this research is to modify the mechanical properties of TCP to achieve the desired degradation rate for the specific need, and improve the in vivo bioactivity for early wound healing by incorporating trace elements such as strontium (Sr2+), magnesium (Mg2+) and silicon (Si4+) as dopants. The hypothesis of this research is that the presence of different trace elements in TCP will influence its phase stability, microstructure, mechanical strength, and both in vitro and in vivo bioactivity.Direct three dimensional printing (3DP) was used to fabricate designed interconnected macroporous pure and doped TCP scaffolds. Microwave sintering as opposed to conventional sintering was also used for better densification and higher mechanical strength. A maximum compressive strength of 10.95 ± 1.28 MPa and 12.01 ± 1.56 MPa were achieved for pure and Sr2+-Mg2+ doped TCP scaffolds with 500 μm designed pores (~400 μm after sintering) sintered in microwave furnace, respectively. Substitution of Mg2+ and Sr2+ into calcium (Ca2+) sites of TCP crystal lattice contributed to phase stability and controlled gradual degradation. On the other hand, Si4+ substitution into phosphorous (P5+) sites destabilized the crystal structure and accelerated degradation of TCP.Interconnected macroporous β-TCP scaffolds facilitated in vivo guided bone tissue regeneration through infiltration of cells and extracellular matrix into the designed pores. Presence of Sr2+, Mg2+ and Si4+ into β-TCP induced increased in vivo early bone formation and better bone remodeling through increased extracellular matrix production such as, collagen and osteocalcin, when tested in rat and rabbit distal femur model. The presence of Si4+ along with Mg2+ induced increased new blood vessel formation. Our results exhibited that Sr2+, Mg2+ and Si4+ doped 3DP TCP scaffolds have strong potential in bone tissue engineering applications for early wound healing

Calcium phosphate ceramic systems in growth factor and drug delivery for bone tissue engineering: a review

Calcium phosphates (CaPs) are the most widely used bone substitutes in bone tissue engineering due to their compositional similarities to bone mineral and excellent biocompatibility. In recent years, CaPs, especially hydroxyapatite and tricalcium phosphate, have attracted significant interest in simultaneous use as bone substitute and drug delivery vehicle, adding a new dimension to their application. CaPs are more biocompatible than many other ceramic and inorganic nanoparticles. Their biocompatibility and variable stoichiometry, thus surface charge density, functionality, and dissolution properties, make them suitable for both drug and growth factor delivery. CaP matrices and scaffolds have been reported to act as delivery vehicles for growth factors and drugs in bone tissue engineering. Local drug delivery in musculoskeletal disorder treatments can address some of the critical issues more effectively and efficiently than the systemic delivery. CaPs are used as coatings on metallic implants, CaP cements, and custom designed scaffolds to treat musculoskeletal disorders. This review highlights some of the current drug and growth factor delivery approaches and critical issues using CaP particles, coatings, cements, and scaffolds towards orthopedic and dental applications

Polycaprolactone-coated 3D printed tricalcium phosphate scaffolds for bone tissue engineering: in vitro alendronate release behavior and local delivery effect on in vivo osteogenesis

The aim of this work was to evaluate the effect of in vitro alendronate (AD) release behavior through polycaprolactone (PCL) coating on in vivo bone formation using PCL-coated 3D printed interconnected porous tricalcium phosphate (TCP) scaffolds. Higher AD and Ca(2+) ion release was observed at lower pH (5.0) than that at higher pH (7.4). AD and Ca(2+) release, surface morphology, and phase analysis after release indicated a matrix degradation dominated AD release caused by TCP dissolution. PCL coating showed its effectiveness for controlled and sustained AD release. Six different scaffold compositions, namely, (i) TCP (bare TCP), (ii) TCP + AD (AD-coated TCP), (iii) TCP + PCL (PCL-coated TCP), (iv) TCP + PCL + AD, (v) TCP + AD + PCL, and (vi) TCP + AD + PCL + AD were tested in the distal femoral defect of Sprague-Dawley rats for 6 and 10 weeks. An excellent bone formation inside the micro and macro pores of the scaffolds was observed from histomorphology. Histomorphometric analysis revealed maximum new bone formation in TCP + AD + PCL scaffolds after 6 weeks. No adverse effect of PCL on bioactivity of TCP and in vivo bone formation was observed. All scaffolds with AD showed higher bone formation and reduced TRAP (tartrate resistant acid phosphatase) positive cells activity compared to bare TCP and TCP coated with only PCL. Bare TCP scaffolds showed the highest TRAP positive cells activity followed by TCP + PCL scaffolds, whereas TCP + AD scaffolds showed the lowest TRAP activity. A higher TRAP positive cells activity was observed in TCP + AD + PCL compared to TCP + AD scaffolds after 6 weeks. Our results show that in vivo local AD delivery from PCL-coated 3DP TCP scaffolds could further induce increased early bone formation

Effect of Chemistry on Osteogenesis and Angiogenesis Towards Bone Tissue Engineering Using 3D Printed Scaffolds

The functionality or survival of tissue engineering constructs depends on the adequate vascularization through oxygen transport and metabolic waste removal at the core. This study reports the presence of magnesium and silicon in direct three dimensional printed (3DP) tricalcium phosphate (TCP) scaffolds promotes in vivo osteogenesis and angiogenesis when tested in rat distal femoral defect model. Scaffolds with three different interconnected macro pore sizes were fabricated using direct three dimensional printing. In vitro ion release in phosphate buffer for 30 days showed sustained Mg  and Si  release from these scaffolds. Histolomorphology and histomorphometric analysis from the histology tissue sections revealed a significantly higher bone formation, between 14 and 20% for 4-16 weeks, and blood vessel formation, between 3 and 6% for 4-12 weeks, due to the presence of magnesium and silicon in TCP scaffolds compared to bare TCP scaffolds. The presence of magnesium in these 3DP TCP scaffolds also caused delayed TRAP activity. These results show that magnesium and silicon incorporated 3DP TCP scaffolds with multiscale porosity have huge potential for bone tissue repair and regeneration

Lovastatin release from polycaprolactone coated β-tricalcium phosphate: Effects of pH, concentration and drug–polymer interactions

The approach of local drug delivery from polymeric coating is currently getting significant attention for both soft and hard tissue engineering applications for sustained and controlled release. The chemistry of the polymer and the drug, and their interactions influence the release kinetics to a great extent. Here, we examine lovastatin release behaviour from polycaprolactone (PCL) coating on β-tricalcium phosphate (β-TCP). Lovastatin was incorporated into biodegradable water insoluble PCL coating. A burst and uncontrolled lovastatin release was observed from bare β-TCP, whereas controlled and sustained release was observed from PCL coating. A higher lovastatin release was observed pH7.4 as compared to pH5.0. Effect of PCL concentration on lovastatin release was opposite at pH7.4 and 5.0. At pH5.0 lovastatin release was decreased with increasing PCL concentration, whereas release was increased with increasing PCL concentration at pH7.4. High Ca2+ ion concentration due to high solubility of β-TCP and degradation of PCL coating were observed at pH5.0 compared to no detectable Ca2+ ion release and visible degradation of PCL coating at pH7.4. The hydrophilic–hydrophobic and hydrophobic–hydrophobic interactions between lovastatin and PCL were found to be the key factors controlling the diffusion dominated release kinetics of lovastatin from PCL coating over dissolution and degradation processes. Understanding the lovastatin release chemistry from PCL will be beneficial for designing drug delivery devices from polymeric coating or scaffolds. [Display omitted] •Lovastatin release chemistry from PCL coated β-TCP was examined.•Incorporation of lovastatin into PCL coating contributed to a controlled release.•PCL amount in the coating and pH of the release media affected the release kinetics.•Drug–polymer hydrophilicity and hydrophobicity were the dominant factors

Electrically polarized biphasic calcium phosphates: adsorption and release of bovine serum albumin

In this study, we applied electrical polarization technique to increase adsorption and control protein release from biphasic calcium phosphate (BCP). Three different biphasic calcium phosphate (BCP) composites, with hydroxyapatite (HAp) and β-tricalcium phosphate (β-TCP), were processed and electrically polarized. Our study showed that stored charge was increased in the composites with the increase in HAp percentage. Adsorption of bovine serum albumin (BSA), as a model protein, on the poled as well as unpoled surfaces of the composites was studied. The highest amount of BSA adsorption was obtained on positively poled surfaces of each composite. Adsorption isotherm study suggested a multilayer adsorption of BSA on the BCP composites. The effect of electrical polarization on BSA release kinetics from positively charged BCP surfaces was studied. A gradual increase in percent BSA release from positively charged BCP surfaces with decreasing stored charge was observed. Our study showed that the BCP based composites have the potential to be used as a drug or growth factor delivery vehicle

Silver doped resorbable tricalcium phosphate scaffolds for bone graft applications

Bone graft procedures, in particular maxillofacial repair, account for half of the orthopedic procedures done in the US each year. Infection is a major issue in surgery, and should be of primary concern when engineering biomaterials. Silver is of renewed importance today, as it has the ability to potentiate antibiotics against resistant bacterial strains. In order to reduce long term infection risks, it is necessary for the scaffold to maintain a silver ion release for the length of the healing process. In this study, silver doped porous β-tricalcium phosphate (β-TCP) scaffolds were engineered using liquid porogen based method with the goal of meeting these requirements. Silver was added to the β-TCP at three different dopant levels: 0.5wt% Ag O, 1wt% Ag O and 2wt% Ag O. Immersion in pH5 acetate buffer over a 60day period resulted in a total cumulative ion release between 32 and 54μM for dense control scaffolds, and between 80 and 90μM for porous scaffolds. Porosity increased the dissolution rate of the scaffolds by a factor of 2. Human osteoblast cell lines were grown on the scaffolds to measure cytotoxicity and cell proliferation. Porosity increased osteoconduction by doubling the cell growth, and there was no significant cytotoxic effect even for the 2wt% Ag O, as cells were observed on all the samples. Our results showed that silver can be released over a long period without compromising the biocompatibility of the scaffolds

Microwave-processed nanocrystalline hydroxyapatite: Simultaneous enhancement of mechanical and biological properties

Despite the excellent bioactivity of hydroxyapatite (HA) ceramics, poor mechanical strength has limited the applications of these materials primarily to coatings and other non-load-bearing areas as bone grafts. Using synthesized HA nanopowder, dense compacts with grain sizes in the nanometer to micrometer range were processed via microwave sintering between 1000 and 1150 °C for 20 min. Here we demonstrate that the mechanical properties, such as compressive strength, hardness and indentation fracture toughness, of HA compacts increased with a decrease in grain size. HA with 168 ± 86 nm grain size showed the highest compressive strength of 395 ± 42 MPa, hardness of 8.4 ± 0.4 GPa and indentation fracture toughness of 1.9 ± 0.2 MPa m 1/2. To study the in vitro biological properties, HA compacts with grain size between 168 nm and 1.16 μm were assessed for in vitro bone cell–material interactions with human osteoblast cell line. Vinculin protein expression for cell attachment and bone cell proliferation using MTT assay showed that surfaces with finer grains provided better bone cell–material interactions than coarse-grained samples. Our results indicate simultaneous improvements in mechanical and biological properties in microwave sintered HA compacts with nanoscale grain size