Musculoskeletal tissue engineering: Adipose derived stromal cell implementation for the treatment of osteoarthritis.

Osteoarthritis (OA) is a progressive degenerative joint disease which results in chronic degeneration of articular cartilage and sclerosis of bone. While tendons and ligaments may heal to a limited extent, articular cartilage has poor intrinsic regenerative potential, and critical-sized bone defects and pathological fractures cannot regenerate spontaneously. OA represents a significant burden of disease globally, affecting 240 million people in the world. The objective of tissue engineering is to recapitulate the natural healing cascade and developmental process by transplanting stromal and progenitor cells which can act directly or indirectly. As the ultimate goal of regenerative medicine is to avoid in vitro expansion of cells and its associated complications, the adipose-derived stromal cell (ASC) is an attractive progenitor cell for tissue engineering for treatment of OA. While clinical studies are still in their infancy, ASCs together with novel scaffold materials represent promising treatment options for patients suffering from OA. How ASCs exert their regenerative potential is a topic of debate, whereby it may be a result of direct differentiation of ASCs into the desired regenerating tissue, and/or through paracrine activity. With the advancement of material science, it is increasingly possible to enhance engraftment of ASCs through the use of biomaterials or to direct progenitor cell fate by activating biophysical signals through designed material microstructures. There are currently over 180 completed or ongoing registered early stage clinical trials involving ASCs, with 17 completed studies reviewed herein detailing the use of ASCs in OA. In order for ASC therapy to become an "off-the-shelf" option for treating OA, several strategies are currently being explored such as ASC cryopreservation and use of allogeneic ASCs. Newer approaches, such as exosome therapy, allow for the use of acellular ASC-derived therapies and are also currently the focus of ongoing investigations.

Stem and progenitor cells: advancing bone tissue engineering.

Unlike many other postnatal tissues, bone can regenerate and repair itself; nevertheless, this capacity can be overcome. Traditionally, surgical reconstructive strategies have implemented autologous, allogeneic, and prosthetic materials. Autologous bone--the best option--is limited in supply and also mandates an additional surgical procedure. In regenerative tissue engineering, there are myriad issues to consider in the creation of a functional, implantable replacement tissue. Importantly, there must exist an easily accessible, abundant cell source with the capacity to express the phenotype of the desired tissue, and a biocompatible scaffold to deliver the cells to the damaged region. A literature review was performed using PubMed; peer-reviewed publications were screened for relevance in order to identify key advances in stem and progenitor cell contribution to the field of bone tissue engineering. In this review, we briefly introduce various adult stem cells implemented in bone tissue engineering such as mesenchymal stem cells (including bone marrow- and adipose-derived stem cells), endothelial progenitor cells, and induced pluripotent stem cells. We then discuss numerous advances associated with their application and subsequently focus on technological advances in the field, before addressing key regenerative strategies currently used in clinical practice. Stem and progenitor cell implementation in bone tissue engineering strategies have the ability to make a major impact on regenerative medicine and reduce patient morbidity. As the field of regenerative medicine endeavors to harness the body's own cells for treatment, scientific innovation has led to great advances in stem cell-based therapies in the past decade.

Impact of surgical innovation on tissue repair in the surgical patient.

BACKGROUND: Throughout history, surgeons have been prolific innovators, which is hardly surprising as most surgeons innovate daily, tailoring their intervention to the intrinsic uniqueness of each operation, each patient and each disease. Innovation can be defined as the application of better solutions that meet new requirements, unarticulated needs or existing market needs. In the past two decades, surgical innovation has significantly improved patient outcomes, complication rates and length of hospital stay. There is one key area that has great potential to change the face of surgical practice and which is still in its infancy: the realm of regenerative medicine and tissue engineering. METHODS: A literature review was performed using PubMed; peer-reviewed publications were screened for relevance in order to identify key surgical innovations influencing regenerative medicine, with a focus on osseous, cutaneous and soft tissue reconstruction. RESULTS: This review describes recent advances in regenerative medicine, documenting key innovations in osseous, cutaneous and soft tissue regeneration that have brought regenerative medicine to the forefront of the surgical imagination. CONCLUSION: Surgical innovation in the emerging field of regenerative medicine has the ability to make a major impact on surgery on a daily basis.

Biomaterials for craniofacial bone engineering.

Conditions such as congenital anomalies, cancers, and trauma can all result in devastating deficits of bone in the craniofacial skeleton. This can lead to significant alteration in function and appearance that may have significant implications for patients. In addition, large bone defects in this area can pose serious clinical dilemmas, which prove difficult to remedy, even with current gold standard surgical treatments. The craniofacial skeleton is complex and serves important functional demands. The necessity to develop new approaches for craniofacial reconstruction arises from the fact that traditional therapeutic modalities, such as autologous bone grafting, present myriad limitations and carry with them the potential for significant complications. While the optimal bone construct for tissue regeneration remains to be elucidated, much progress has been made in the past decade. Advances in tissue engineering have led to innovative scaffold design, complemented by progress in the understanding of stem cell-based therapy and growth factor enhancement of the healing cascade. This review focuses on the role of biomaterials for craniofacial bone engineering, highlighting key advances in scaffold design and development.

Immunomodulatory effects of a traditional Chinese medicine with potential antiviral activity: a self-control study.

Traditional Chinese medicine (TCM) has been used for prevention and treatment of severe acute respiratory syndrome (SARS) in Hong Kong during the outbreak in spring 2003. We investigated the immunomodulating effects of an innovative TCM regimen derived from two herbal formulas (Sang Ju Yin and Yu Ping Feng San) for treating febrile diseases. Thirty-seven healthy volunteers were given the oral TCM regimen daily for 14 days. Peripheral venous blood samples were taken on days 0, 15 and 29 for hematology, biochemistry and immunology tests, including the measurement of blood lymphocyte subsets and plasma T-helper lymphocyte types 1 and 2 cytokines and receptor. After 3 months, 23 of the volunteers participated in a control study without TCM treatment for the same time course of blood tests. Two volunteers withdrew on day 2, due to headache and dizziness. All others remained well without any side effects. No participants showed significant changes in their blood test results, except that the T-lymphocyte CD4/CD8 ratio increased significantly from 1.31 +/- 0.50 (mean +/- SD) on day 0 to 1.41 +/- 0.63 on day 15 (p < 0.02), and reduced to 1.32 +/- 0.47 on day 29 (p < 0.05). In the control study, there were no changes in the CD4/CD8 ratio. The transient increase in CD4/CD8 ratio was likely due to the TCM intake. We postulate that the administration of the innovative TCM may have beneficial immunomodulatory effects for preventing viral infections including SARS.

Inhibitory effects of antifungal proteins on human immunodeficiency virus type 1 reverse transcriptase, protease and integrase.

A variety of antifungal proteins were isolated from seeds of leguminous plants including French bean, cowpea, field bean, mung bean, peanut and red kidney bean. They were assayed for ability to inhibit human immunodeficiency virus type I (HIV-1) reverse transcriptase, protease and integrase, enzymes essential to the life cycle of HIV-1 . It was found that the cowpea beta-antifungal protein had a high potency in inhibiting HIV-1 protease and HIV-1 integrase. Cowpea alpha-antifungal protein was potent in inhibiting HIV-1 reverse transcriptase and HIV-1 integrase. Peanut antifungal protein was characterized by a high inhibitory activity against HIV-1 integrase and an intermediate potency in inhibiting HIV- I reverse transcriptase and HIV- I protease. French bean thaumatin-like protein expressed low HIV- I protease inhibitory activity and red kidney bean lectin inhibited HIV- I integrase by only a very small extent. Antifungal proteins from the field bean and mung bean had an intermediate potency in inhibitory HIV-1 protease and integrase. However, mung bean antifungal protein was not capable of inhibiting HIV-1 reverse transcriptase. The results indicate that nearly all leguminous antifungal proteins examined were able to inhibit HIV-1 reverse transcriptase, protease and integrase to some extent.

The cyclic AMP-mediated expression of acetylcholinesterase in myotubes shows contrasting activation and repression between avian and mammalian enzymes.

Cyclic adenosine 3',5'-monophosphate (cAMP)-dependent signalling pathway has been proposed to regulate acetylcholinesterase (AChE) expression in chick muscle; however, its role in mammalian enzyme is not known. We provide several lines of evidence to suggest that the cAMP-mediated AChE expression in myotube is oppositely regulated between avian and mammalian enzymes. Human AChE promoter was tagged with luciferase, namely Hp-Luc, which was transfected into cultured chick myotubes. Application of cAMP and forskolin induced the expression of chick AChE but reduced human AChE promoter-driven luciferase activity. Transfection of cDNAs encoding active mutants of G proteins altered the intracellular cAMP level in myotubes as well as the expression of chick and human AChE. When the constitutively active forms of Activating Transcription Factor-1 (EWS/ATF-1 oncogene) were over expressed in Hp-Luc transfected myotubes, the expression of chick AChE transcript and protein increased from approximately 1.8- to approximately 2.5-fold, but the luciferase activity was decreased by over 60%. Overexpression of cAMP-responsive element binding protein (CREB) in Hp-Luc transfected myotubes markedly enhanced the cAMP-mediated AChE expression in up- and downregulated chick and human enzymes, respectively. In addition, CREB bound the CRE sequence of human AChE promoter. Mutation on the CRE site markedly enhanced the expression of the promoter-driven luciferase; however, its response to cAMP inhibition in cultured myotubes was still retained. These findings suggest that a cAMP-dependent pathway is contrasting activation and repression of AChE expression in chick and human muscles.

A comparison of HIV-1 integrase inhibition by aqueous and methanol extracts of Chinese medicinal herbs.

The aqueous and methanol extracts of twenty herbs traditionally used in Chinese medicine were screened for anti-HIV-1 integrase activity in a non-radioactive ELISA-based HIV-1 integrase assay. The screening was performed at an herb extract concentration of 50 microg/ml. It was found that most of the aqueous and methanol herb extracts could elicit strong inhibition of HIV-I integrase activity. The inhibition was most likely due to tannins or polyphenolics in the herb extracts. In most of the herb extracts, 40-80% of the anti-HIV-1 integrase activity could be removed after passing through a minicolumn of polyamide resin. After removal of polyphenolic compounds, the methanol extract of Paeonia suffruticosa still exerted potent inhibition of HIV-1 integrase (EC50 = 15 microg/ml) and the aqueous extract of Prunella vulgaris caused moderate inhibition (EC50 = 45 microg/ml). The results support the view that herbs represent a rich source of anti-HIV compounds.

A comparison of human immunodeficiency virus type-1 protease inhibition activities by the aqueous and methanol extracts of Chinese medicinal herbs.

The aqueous and methanol extracts of thirty-one herbs traditionally used as anti-fever remedies in China were screened for their in vitro inhibition on human immunodeficiency virus type-1 protease (HIV-1 PR). The activity of recombinant HIV-1 protease was determined by sequence-specific cleavage at the Tyr-Pro bond of the fluorogenic substrate (Arg-Glu(EDANS)-Ser-Gln-Asn-Tyr-Pro-Ile-Val-Gln-Lys(DABCYL)- Arg) or by HPLC anaylsis of the cleavage products after incubation of the enzyme with a synthetic peptide substrate (Acetyl-Ser-Gln-Asn-Tyr-Pro-Val-Val-amide). Among the herbal extracts examined, the aqueous extracts of Prunella vulgaris and Scutellaria baicalensis and the methanol extracts of Woodwardia unigemmata, Paeonica suffruticosa and Spatholobus suberectus elicited significant inhibition (>90%) at a concentration of 200 microg/ml.

The promoter of human acetylcholinesterase is activated by a cyclic adenosine 3',5'-monophosphate-dependent pathway in cultured NG108-15 neuroblastoma cells.

Different transcription elements have been proposed to play a role in the regulation of acetylcholinesterase (AChE) in muscle and neuron, and cyclic adenosine 3',5'-monophosphate (cAMP)-dependent pathway is one of them. In order to test the possible role of cAMP in regulating the expression of human AChE, an approximately 2.2 kb DNA fragment of human AChE promoter was linked up stream to a luciferase reporter. The chimeric DNA was transfected into cultured NG108-15 neuroblastoma cells. Application of Bt(2)-cAMP and forskolin increased the promoter driven luciferase activity over 2-fold in the transfected NG108-15 cells; the increase was parallel to the activation of endogenous AChE protein and enzymatic activity. The intracellular cAMP level was increased in the Galpha(sQL) (constitutively active mutant of Galpha(s)) cDNA transfected NG108-15 cells. The Galpha(sQL) cDNA transfected cells showed an increase of over 10-fold in the luciferase activity. In addition, a constitutively active mutant of activating transcription factor-1 (ATF-1) was able to turn on human AChE promoter by approximately 4-fold when they were co-expressed in the neuroblastoma cells. These results support the involvement of a cAMP-dependent pathway in regulating the expression of human AChE.

The plant ribosome inactivating proteins luffin and saporin are potent inhibitors of HIV-1 integrase.

The ribosome inactivating proteins (RIPs) are a group of proteins that are able to inactivate eukaryotic protein synthesis by attacking the 28S ribosomal RNA. Recent studies have shown that some RIPs possess strong anti-human immunodeficiency virus (HIV) activity. In this study, several common plant RIPs including agrostin, gelonin, luffin, alpha-momorcharin, beta-momorcharin, saporin and trichosanthin were examined for the ability to interfere with HIV-1 replication in a variety of mechanistic assays in vitro. These assays included the CD4/gp120 interaction assay, HIV-1 reverse transcriptase (RT) assay, HIV-1 protease assay and HIV-1 integrase assay. At the concentration of 100 nM, all RIPs appeared to enhance the CD4/gp120 interaction by about 50%. These RIPs exhibited a very weak suppressive effect on HIV-1 RT and on HIV-1 protease. In contrast, with the exception of agrostin, all the RIPs tested could strongly inhibit HIV-1 integrase, the extent of inhibition ranging from 26.1 to 96.3% in an ELISA-based assay. Two RIPs, saporin and luffin, which licited over 90% inhibition in the ELISA-based assay, were further characterized in a radiometric assay. Both of these two RIPs evoked a strong dose-dependent inhibition in the 3'-end processing and strand-transfer activities of integrase. The results from this study suggest that the anti-HIV property of RIPs may be due to inhibition of HIV-1 integrase.

Characterization of a functional AT1A angiotensin receptor in pancreatoma AR4-2J cells.

Functional angiotensin receptors were characterized in the rat pancreatic acinar cell line AR4-2J. Angiotensin II stimulated a dose-dependent release of amylase and production of inositol phosphates. Results of high-performance liquid chromatography separation of inositol phosphates indicated that angiotensin stimulated the rapid accumulation of inositol 1,3,4-trisphosphate. Angiotensin II and angiotensin III were at least an order of magnitude more potent than angiotensin I in the stimulation of amylase release. The angiotensin II-stimulated amylase release was blocked by losartan, a selective AT1 angiotensin antagonist. The selective AT2 angiotensin receptor ligands CGP42112 did not alter angiotensin II-stimulated amylase released. However, CGP42112 stimulated amylase release at micromolar concentrations with a potency similar to angiotensin I. Analysis of mRNA expression by reverse transcription polymerase chain reaction suggested that AT1A was the predominant type-I angiotensin receptor expressed in the AR4-2J cells.

Lack of allelic association of dopamine D4 receptor gene polymorphisms with Parkinson's disease in a Chinese population.

Parkinson's disease (PD) is a neurodegenerative disease caused by a multitude of environmental, neurochemical, and genetic factors. The gene for human dopamine D4 receptor (DRD4) has been considered as a plausible candidate for the pathogenesis of PD. Different dopamine D4 receptor allelic forms have variable affinity toward certain neuroleptics such as clozapine, suggesting a role for dopamine D4 receptors in neurologic disorders. To test the hypothesis that the DRD4 polymorphism is associated with the susceptibility to Parkinson's disease, we have examined differences in allele frequencies of different DRD4 polymorphisms in 101 Chinese patients with PD and in 105 age-matched control subjects in Hong Kong. The DRD4 gene was analyzed by a non-radioactive polymerase chain reaction-based Southern hybridization with chemiluminescence detection. The number of polymorphic 48 base pair tandem repeats in exon 3 was identified in each study subject. The DRD4 alleles with high frequencies in the control subjects are 4-repeat allele (72.4%), 2-repeat allele (21.4%), and 7-repeat allele (3.8%) which accounted for over 97% of the total alleles in the elderly Chinese population. The most prevalent genotype in the control subjects is the 4/4 (47.6%), followed by 4/2 (38.6), 4/7 (7.6%), and 2/2 (3.0%). None of the variable number tandem repeat polymorphism showed evidence for genetic association with Parkinson's disease.

The calcitonin gene-related peptide-induced acetylcholinesterase synthesis in cultured chick myotubes is mediated by cyclic AMP.

In vertebrate neuromuscular junctions, post-synaptic specialization includes aggregation of acetylcholine receptors (AChRs) and acetylcholinesterase (AChE). The motor nerve provides soluble factors and electrical activity to achieve this striking localization of AChRs/AChE. Calcitonin gene-related peptide (CGRP), a neuropeptide synthesized by motor neurons, is able to stimulate the expression of AChR in cultured myotubes. Similar to AChR regulation, synthesis of AChE in cultured chick myotubes is also stimulated by CGRP. Application of CGRP onto cultured myotubes stimulated the accumulation of intracellular cyclic AMP (cAMP) as well as the expression of AChE mRNA and protein. However, the enzymatic activity of AChE remained unchanged. In cultured myotubes, various drugs affecting the intracellular level of cAMP, such as N6,O2'-dibutyryladenosine 3',5'-cyclic monophosphate, cholera toxin, and forskolin, could mimic the effect of CGRP in stimulating the expression of AChE. When myotubes were transfected with cDNA encoding constitutively active mutant Galpha(s), the intracellular cAMP synthesis was increased. The increase in cAMP level was in parallel with an increase in the expression of AChE, whereas transfection of active mutant Galpha(i) cDNA decreased the cAMP level as well as the AChE expression. In addition, expression of collagen-tailed AChE was up-regulated by the cAMP pathway. These findings indicated that CGRP-induced AChE regulation is mediated by the cAMP pathway and represented the first evidence to suggest that the regulation of mRNA synthesis of AChR and AChE can be mediated by the same neuron-derived factor.

Over-expression of acetylcholinesterase stimulates the expression of agrin in NG108-15 cells.

Several lines of evidence suggest the non-cholinergic functions of acetylcholinesterase (AChE) in promoting neurite outgrowth of cultured neurons and in inducing the postsynaptic specializations of developing neuromuscular junctions. In order to support the hypothesis, a cholinergic synapse-forming cell line NG108-15 was over-expressed with chick AChE by cDNA transfection. The transfected NG108-15 cells secreted a approximately 105-kDa protein, recognized by anti-AChE antibody in Western blot analysis, corresponding to the chick AChE catalytic subunit. Over 80% of the recombinant enzyme were secreted into the conditioned medium and they were enzymatically active. In the NG108-15 cell-muscle co-cultures, the AChR-aggregating activity of NG108-15 cells was increased by the over-expression of AChE. The increase in AChR-aggregating activity of the transfected NG108-15 cells paralleled with the increase in agrin and neurofilament expression of the transfected cells as determined by their corresponding antibodies. However, the intracellular cAMP level remained unchanged in the AChE over-expressed NG108-15 cells. These results support the hypothesis that AChE could play a role in promoting neuron differentiation.

NG108-15 cells induce the expression of muscular acetylcholinesterase when co-cultured with myotubes.

Although muscular activity has been demonstrated to regulate the expression of acetylcholinesterase (AChE) in cultured myotubes, the exact role of the presynaptic terminus in regulating AChE expression at the neuromuscular junctions is not known. A chimeric co-culture of neuroblastoma x glioma hybrid NG108-15 cells with chick myotubes was established. By using chick-specific anti-AChE antibody, a protein of approximately 105 kDa in size corresponding to chick AChE catalytic subunit was revealed by Western blot analysis from the extracts of neuron-muscle co-cultures. In the co-cultures, NG108-15 cells induced the up regulation of muscle AChE expression by approximately 2.5-fold, while the control protein, chick muscle alpha-actinin at approximately 100 kDa, remained relatively unchanged. The NG108-15 cell-induced muscle AChE expression in the co-cultures was persistent when the muscular activity was blocked by alpha-bungarotoxin. In order to determine the AChE-inducing activity derived from NG108-15 cells, the cultured chick myotubes were treated with either conditioned medium of NG108-15 cells or its cell lysate. However, the muscle AChE, both in protein and activity levels, remained relatively unchanged. Our finding suggests that an AChE-inducing factor(s) is derived from the neuroblastoma cells in the co-cultures, but that may require the nerve-muscle contacts in culture.

Denervation decreases the ipsilateral expression of AChE in chick lumbaric motor neurons.

In vertebrate neuromuscular junctions, acetylcholinesterase (AChE; EC 3.1.1.7) is highly concentrated at the synaptic basal lamina and the postsynaptic muscle fiber. The postsynaptic muscle cell is the primary source of AChE. However, several lines of evidence indicate that the presynaptic motor neuron is able to synthesize and secrete AChE at the neuromuscular junctions. By using anti-AChE monoclonal antibody in immunohistochemical staining, we found that the AChE-positive cells were labeled only at the motor neurons of the chick spinal cords. When the protein extract of chick spinal cords was analyzed by a Western blot analysis, a protein band of approximately 105 kDa was recognized. In denervated chicks, the expression of motor neuron AChE, as recognized on a Western blot, decreased by approximately 50% 4 days after denervation. The AChE expression in denervated chick spinal cords, however, was restored to control level 10 days after denervation. The decreased AChE expression was restricted to the ipsilateral side of the denervated chick spinal cord while the contralateral side was relatively unchanged. In comparison with the contralateral side, the level of AChE protein and enzymatic activity expressed in the ipsilateral spinal cord was approximately 50% lower. This is the first demonstration to show that the ipsilateral and contralateral sides of chick spinal cords respond differently after nerve denervation.

A globular, not asymmetric, form of acetylcholinesterase is expressed in chick motor neurons: down-regulation toward maturity and after denervation.

In vertebrate neuromuscular junctions, the postsynaptic specializations include the accumulation of acetylcholinesterase (AChE) at the synaptic basal lamina and the muscle fiber. Several lines of evidence indicate that the presynaptic motor neuron is able to synthesize and secrete AChE at the neuromuscular junctions. By using anti-AChE catalytic subunit, anti-butyrylcholinesterase (BuChE) catalytic subunit, and anti-AChE collagenous tail monoclonal antibodies, we demonstrated that the motor neurons of chick spinal cord expressed AChE in vivo and the predominant AChE was the globular form of the enzyme. Neither asymmetric AChE nor BuChE was detected in the motor neurons. The molecular mass of AChE catalytic subunit in the motor neuron was approximately 105 kDa, which was similar to that of the globular enzyme from low-salt extracts of muscle; both of them were approximately 5 kDa smaller than the asymmetric AChE from high-salt extracts of muscle. The level of AChE expression in the motor neurons decreased, as found by immunochemical and enzymatic analysis, during the different stages of the chick's development and after nerve lesion. Thus, the AChE activity at the neuromuscular junctions that is contributed by the presynaptic motor neurons is primarily the globular, not the asymmetric, form of the enzyme, and these contributions decreased toward maturity and after denervation.