Alfred C. Kuo

Transmembrane transforming growth factor-α tethers to the PDZ domain-containing, Golgi membrane-associated protein p59/GRASP55

Transforming growth factor‐α (TGF‐α) and related proteins represent a family of transmembrane growth factors with representatives in flies and worms. Little is known about the transport of TGF‐α and other transmembrane growth factors to the cell surface and its regulation. p59 was purified as a cytoplasmic protein, which at endogenous levels associates with transmembrane TGF‐α. cDNA cloning of p59 revealed a 452 amino acid sequence with two PDZ domains. p59 is myristoylated and palmitoylated, and associates with the Golgi system, where it co‐localizes with TGF‐α. Its first PDZ domain interacts with the C‐terminus of transmembrane TGF‐α and select transmembrane proteins. p59 is the human homolog of GRASP55, which is structurally related to GRASP65. GRASP55 and GRASP65 have been shown to play a role in stacking of the Golgi cisternae in vitro. C‐terminal mutations of transmembrane TGF‐α, which decrease or abolish the interaction with p59, also strongly impair cell surface expression of TGF‐α. Our observations suggest a role for membrane tethering of p59/GRASP55 to select transmembrane proteins, including TGF‐α, in maturation and transport to the cell surface.

Structured three-dimensional co-culture of mesenchymal stem cells with chondrocytes promotes chondrogenic differentiation without hypertrophy

OBJECTIVE This study investigated a novel approach to induce chondrogenic differentiation of human mesenchymal stem cells (hMSC). We hypothesized that a structured three-dimensional co-culture using hMSC and chondrocytes would provide chondroinductive cues to hMSC without inducing hypertrophy. METHOD In an effort to promote optimal chondrogenic differentiation of hMSC, we created bilaminar cell pellets (BCPs), which consist of a spherical population of hMSC encased within a layer of juvenile chondrocytes (JC). In addition to histologic analyses, we examined proteoglycan content and expression of chondrogenic and hypertrophic genes in BCPs, JC pellets, and hMSC pellets grown in the presence or absence of transforming growth factor-β (TGFβ) following 21 days of culture in either growth or chondrogenic media. RESULTS In either growth or chondrogenic media, we observed that BCPs and JC pellets produced more proteoglycan than hMSC pellets treated with TGFβ. BCPs and JC pellets also exhibited higher expression of the chondrogenic genes Sox9, aggrecan, and collagen 2A1, and lower expression of the hypertrophic genes matrix metalloproteinase-13, Runx2, collagen 1A1, and collagen 10A1 than hMSC pellets. Histologic analyses suggest that JC promote chondrogenic differentiation of cells in BCPs without hypertrophy. Furthermore, when cultured in hypoxic and inflammatory conditions intended to mimic the injured joint microenvironment, BCPs produced significantly more proteoglycan than either JC pellets or hMSC pellets. CONCLUSION The BCP co-culture promotes a chondrogenic phenotype without hypertrophy and, relative to pellet cultures of hMSCs or JCs alone, is more resistant to the adverse conditions anticipated at the site of articular cartilage repair.

The tissue diagnostic instrument

Tissue mechanical properties reflect extracellular matrix composition and organization, and as such, their changes can be a signature of disease. Examples of such diseases include intervertebral disk degeneration, cancer, atherosclerosis, osteoarthritis, osteoporosis, and tooth decay. Here we introduce the tissue diagnostic instrument (TDI), a device designed to probe the mechanical properties of normal and diseased soft and hard tissues not only in the laboratory but also in patients. The TDI can distinguish between the nucleus and the annulus of spinal disks, between young and degenerated cartilage, and between normal and cancerous mammary glands. It can quantify the elastic modulus and hardness of the wet dentin left in a cavity after excavation. It can perform an indentation test of bone tissue, quantifying the indentation depth increase and other mechanical parameters. With local anesthesia and disposable, sterile, probe assemblies, there has been neither pain nor complications in tests on patients. We anticipate that this unique device will facilitate research on many tissue systems in living organisms, including plants, leading to new insights into disease mechanisms and methods for their early detection.

Comorbidities and Perioperative Complications in HIV-Positive Patients Undergoing Primary Total Hip and Knee Arthroplasty

BACKGROUND Highly active antiretroviral therapy has prolonged the lifespan of individuals infected with human immunodeficiency virus (HIV). We hypothesized that the number of primary total joint arthroplasties performed in this population has been increasing and that HIV infection is not an independent risk factor for postoperative complications. METHODS The Nationwide Inpatient Sample for the years 2000 through 2008 was queried to identify patients who underwent primary total joint arthroplasty. HIV, comorbidities, and complications were identified with use of ICD-9-CM (International Classification of Diseases, Ninth Revision, Clinical Modification) codes. Data were analyzed with use of multivariate logistic regression, the Pearson chi-square test, and the Mann-Kendall trend test. RESULTS Of the estimated 5,681,024 admissions for primary total hip and knee arthroplasty in the United States during this period, 8229 (0.14%) were in patients who had HIV. Compared with HIV-negative patients (controls), infected patients were more likely to be younger, be male, and have a history of osteonecrosis, liver disease, drug use, and coagulopathy. The number of total hip and total knee arthroplasties in HIV-positive patients increased from 2000 to 2008 (p < 0.05). Seventy-nine percent (6499) of the total joint arthroplasties in the HIV-positive patients involved the hip. Compared with HIV-negative patients undergoing total hip arthroplasty, HIV-positive patients were more likely to develop acute renal failure (1.3% compared with 0.8%, p = 0.04), develop a wound infection (0.6% compared with 0.3%, p = 0.02), and undergo postoperative irrigation and debridement (0.2% compared with 0.1%, p = 0.01). They were less likely to have a myocardial infarction (0.4% compared with 0.9%, p = 0.04). There was no difference in total complications (8.3% compared with 7.8%, p = 0.52). Similarly, there was no difference in total complications in patients undergoing total knee arthroplasty (7.8% compared with 8.0%, p = 0.76). HIV was not an independent risk factor for complications in total hip arthroplasty (odds ratio [OR], 1.18; 95% confidence interval [CI], 0.95 to 1.47) or total knee arthroplasty (OR, 0.78; 95% CI, 0.49 to 1.25). CONCLUSIONS The incidence of primary total joint arthroplasty in HIV-positive patients has been increasing. These patients were at slightly higher risk of certain immediate postoperative complications because of a higher rate of medical comorbidities. HIV infection was not an independent risk factor for the total rate of perioperative complications. LEVEL OF EVIDENCE Prognostic Level II. See Instructions for Authors for a complete description of levels of evidence.

Complications of Morbid Obesity in Total Joint Arthroplasty: Risk Stratification Based on BMI

This study stratifies complication risk in primary total joint arthroplasty (TJA) based on body mass index (BMI). Demographics, co-morbidities, perioperative variables, and complications were reviewed for 22,808 patients. Chi-squared, one-way ANOVA, univariate and multivariable regression analysis were performed. Increasing BMI led to an increase (P<0.05) in combined complications, acute kidney injury (AKI), cardiac arrest (CA), reintubation, reoperation, and superficial infection (SI). Univariate analysis for BMI>40 revealed an increase in combined complications (15.21-vs-17.40%), AKI (1.93-vs-3.87%), CA (0.22-vs-0.57%), reintubation (0.47-vs-0.95%), reoperation (2.36-vs-3.37%), and SI (0.82-vs-1.65%). Multivariable regression showed BMI>40 as an independent predictor for combined complications (OR=1.18), AKI (OR=1.79), CA (OR=3.94), reintubation (OR=2.56), reoperation (OR=1.44), and SI (OR=2.11). Morbid obesity confers increased risk for complications in TJA.

Cytotoxicity of local anesthetics on human mesenchymal stem cells.

BACKGROUND Local anesthetics are frequently delivered intra-articularly to provide perioperative pain control. Previous studies have shown that the commonly used drugs lidocaine, ropivacaine, and bupivacaine can be toxic to human chondrocytes. The present study was conducted to determine whether the toxic effects of local anesthetics on human chondrocytes also extend to human mesenchymal stem cells. METHODS Human mesenchymal stem cells from three healthy donors were grown in tissue culture and exposed to the following anesthetic treatments for sixty minutes: (1) 1% lidocaine, (2) 2% lidocaine, (3) 0.25% bupivacaine, (4) 0.5% bupivacaine, (5) 0.2% ropivacaine, and (6) 0.5% ropivacaine. The cells were then allowed to recover for twenty-four hours in regular growth media, and viability was measured with use of fluorescent staining for live cells or a luminescence assay for ATP content. RESULTS The live cell counts and ATP content were correlated (r2 = 0.79), and 2% lidocaine was found to be significantly more toxic than all doses of bupivacaine and ropivacaine. Treatment with 1% lidocaine resulted in significantly fewer live cells (49%) compared with the control, and the live cell count was also significantly less than that for the other anesthetics. However, the ATP level in the 1% lidocaine group was not significantly lower than those in the other groups. Bupivacaine and ropivacaine did not exhibit significant differences in toxicity compared with the control or with each other. CONCLUSIONS Ropivacaine and bupivacaine had limited toxicity in human mesenchymal stem cells. However, lidocaine could significantly decrease mesenchymal stem cell viability. Since other studies have shown ropivacaine to be less toxic to chondrocytes than bupivacaine, ropivacaine may be a safer intra-articular anesthetic. CLINICAL RELEVANCE Mesenchymal stem cells likely play a key role in healing following surgical procedures such as microfracture and ligament reconstruction. If local anesthetics are used following joint surgery, selection of an agent with low toxicity toward mesenchymal stem cells, such as ropivacaine, may maximize tissue healing potential.

Comparison of gene expression patterns in articular cartilage and dedifferentiated articular chondrocytes

During monolayer culture, articular chondrocytes dedifferentiate into fibroblast‐like cells. The mechanisms underlying this process are poorly understood. We sought to further characterize dedifferentiation by identifying an extended panel of genes that distinguish articular cartilage from dedifferentiated chondrocytes. Thirty‐nine candidate marker‐genes were identified from previous studies on articular‐cartilage gene‐expression. Real‐time PCR was used to evaluate the mRNA levels for these candidates in calf articular cartilage and dedifferentiated articular chondrocytes. Twenty‐two of the candidate marker genes exhibited at least a two‐fold difference in gene expression in the two cell types. Twelve of these genes had at least a ten‐fold difference in gene expression. Tenascin C (TNC), type I collagen (COL1A1), and hypoxia‐inducible factor 1 alpha (HIF1α) showed the highest relative expression levels in dedifferentiated chonodrocytes. Type II collagen (COL2A1), type XI collagen (COL11A2), and superficial zone protein (SZP) showed the highest relative expression levels in articular cartilage. In contrast to previous findings, fibromodulin mRNA, and protein levels were higher in dedifferentiated chondrocytes. Compared to smaller subsets of markers, this panel of 12 highly differentially expressed genes may more precisely distinguish articular cartilage from dedifferentiated chondrocytes. Since many of the genes up‐regulated in dedifferentiated chondrocytes are also expressed during cartilage development, dedifferentiated chondrocytes may possess features of cartilage precursor cells.

Mid-Term Outcomes in HIV-Positive Patients After Primary Total Hip or Knee Arthroplasty

We hypothesized that infection rates following total joint arthroplasty (TJA) in those with the human immunodeficiency virus (HIV) without hemophilia or drug use would be similar to rates in HIV-negative patients. Records at an urban HIV referral hospital were searched for patients who underwent primary total hip and knee arthroplasty from 2003 to 2010. The primary outcome was revision for infection. 372 HIV-negative and 22 HIV-positive TJA patients met inclusion criteria. The HIV-positive group had more deep infections than the HIV-negative group (9.1% v 2.2%, P=0.102). There were no infections in those with AIDS-defining CD4 counts. Those with HIV may have a higher risk of developing a deep infection. A low CD4 count is not an absolute contraindication to TJA in HIV positive patients.

Trophic stimulation of articular chondrocytes by late‐passage mesenchymal stem cells in coculture

Coculture of mesenchymal stem cells (MSCs) with articular chondrocytes (ACs) increases glycosaminoglycan (GAG) accumulation compared to monoculture. MSCs might (1) differentiate into ACs (progenitor role) and/or (2) stimulate AC matrix metabolism (trophic role). MSCs lose the ability to undergo chondrogenesis after extended passaging. We hypothesized that MSCs act predominantly as progenitors, and that late‐passage MSCs without chondrogenic potential would be unable to increase GAG in coculture. Early‐ and late‐passage human MSCs (hMSCs) were grown in pellet monoculture under chondrogenic conditions and in pellet coculture with bovine ACs. Chondrogenesis was assessed with GAG quantification, safranin‐O staining, and quantitative PCR (qPCR). Contributions of human and bovine cells were assessed with species‐specific qPCR and human‐specific immunostaining. Late‐passage hMSCs did not undergo chondrogenesis in monoculture with chondrogenic stimuli or in coculture with ACs. Early‐passage hMSCs underwent chondrogenesis only in response to chondrogenic stimuli. Coculture pellets in both cases accumulated as much GAG/DNA as monoculture AC pellets. Aggrecan transcription was not increased in coculture. Late‐passage hMSCs that do not undergo chondrogenesis are capable of stimulating GAG accumulation in coculture with ACs. This supports a trophic effect of hMSCs on ACs. hMSCs may have therapeutic utility even after prolonged passaging. Published 2013 by Wiley Periodicals, Inc. on behalf of the Orthopaedic Research Society. J Orthop Res 31:1936–1942, 2013

Inducible immortality in hTERT-human mesenchymal stem cells

Human mesenchymal stem cells (hMSCs) are attractive candidates for tissue engineering and cell‐based therapy because of their multipotentiality and availability in adult donors. However, in vitro expansion and differentiation of these cells is limited by replicative senescence. The proliferative capacity of hMSCs can be enhanced by ectopic expression of telomerase, allowing for long‐term culture. However, hMSCs with constitutive telomerase expression demonstrate unregulated growth and even tumor formation. To address this problem, we used an inducible Tet‐On gene expression system to create hMSCs in which ectopic telomerase expression can be induced selectively by the addition of doxycycline (i‐hTERT hMSCs). i‐hTERT hMSCs have inducible hTERT expression and telomerase activity, and are able to proliferate significantly longer than wild type hMSCs when hTERT expression is induced. They stop proliferating when hTERT expression is turned off and can be rescued when expression is re‐induced. They retain multipotentiality in vitro even at an advanced age. We also used a selective inhibitor of telomere elongation to show that the mechanism driving immortalization of hMSCs by hTERT is dependent upon maintenance of telomere length. Thanks to their extended lifespan, preserved multipotentiality and controlled growth, i‐hTERT hMSCs may prove to be a useful tool for the development and testing of novel stem cell therapies.