From Medicine and Science in Sports and Exercise®

Association Between Physical Activity and Kidney Function

National Health and Nutrition Examination Survey

Marquis S. Hawkins; Mary Ann Sevick; Caroline R. Richardson; Linda F. Fried; Vincent C. Arena; Andrea M. Kriska

Authors and Disclosures

Posted: 08/10/2011; Medicine and Science in Sports and Exercise®. 2011;43(8):1457-1464. © 2011 American College of Sports Medicine

Abstract and Introduction

Abstract

Hawkins, M. S., M. A. Sevick, C. R. Richardson, L. F. Fried, V. C. Arena, and A. M. Kriska. Association between Physical Activity and Kidney Function: National Health and Nutrition Examination Survey.Med. Sci. Sports Exerc., Vol. 43, No. 8, pp. 1457–1464, 2011.Introduction: Chronic kidney disease is a condition characterized by the deterioration of the kidney's ability to remove waste products from the body. Although treatments to slow the progression of the disease are available, chronic kidney disease may eventually lead to a complete loss of kidney function. Previous studies have shown that physical activities of moderate intensity may have renal benefits. Few studies have examined the effects of total movement on kidney function. The purpose of this study was to determine the association between time spent at all levels of physical activity intensity and sedentary behavior and kidney function.
Methods: Data were obtained from the 2003–2004 and 2005–2006 National Health and Nutrition Examination Survey, a cross-sectional study of a complex, multistage probability sample of the US population. Physical activity was assessed using an accelerometer and questionnaire. Glomerular filtration rate (eGFR) was estimated using the Modification of Diet in Renal Disease study formula. To assess linear associations between levels of physical activity and sedentary behavior with log-transformed estimated GFR (eGFR), linear regression was used.
Results: In general, physical activity (light and total) was related to log eGFR in females and males. For females, the association between light and total physical activity with log eGFR was consistent regardless of diabetes status. For males, the association between light and total physical activity and log eGFR was only significant in males without diabetes.
Conclusions: When examining the association between physical activity, measured objectively with an accelerometer, and kidney function, total and light physical activities were found to be positively associated with kidney function.

Introduction

Chronic kidney disease (CKD) is the progressive deterioration of the kidney's ability to remove fluid and nitrogenous waste from the body as well as some other key functions. Diabetes and hypertension are the most common causes of CKD.^[35] Although treatments to slow the progression of the disease are available, CKD may eventually lead to renal failure or end-stage renal disease (ESRD), which is the complete loss of kidney function. Once kidneys have failed, the only available treatment options are dialysis or kidney transplant.

CKD is of immense public health concern and is expected to become a bigger problem in the coming decades. A study of the US population using the National Health and Nutrition Examination Survey (NHANES) estimates that from 1999 to 2004, approximately 17% of adults older than 20 yr had CKD, which is an increase of approximately 16% compared with NHANES data from 1988 to 1994.^[8,28] The prevalence of ESRD is growing as well, doubling since the early 1990s and is projected to reach 2 million by the year 2030.^[32,34] The growing prevalence of CKD is alarming considering the economic and physical burdens it places on the individual as well as the health care system.^[22]

Glomerular filtration rate (GFR), which is a measure of the kidney's ability to clear plasma of nitrogenous wastes over a specific amount of time, is regarded as the best way to assess kidney function. In clinical practice and epidemiologic studies, GFR is estimated (eGFR) using a formula based on serum creatinine, gender, race, and age. A reduction in GFR, along with other evidence of kidney damage, is in part used to classify severity/stages of kidney dysfunction. A recent analysis from the ATTICA study indicated that individuals with moderate to severe kidney dysfunction had more than three times the risk of developing a cardiovascular disease (CVD) during the 5-yr study follow-up compared with individuals with normal kidney function.^[7] The Atherosclerosis Risk in Communities (ARIC) Study was a prospective population-based study that examined the relationship between change in eGFR with CHD and all-cause mortality in 13,029 white and black men and women between the ages of 45 and 64 yr. The study showed that individuals with annual declines in eGFR ≥5% had the highest risk for CHD and all-cause mortality compared with individuals with annual declines in eGFR of <5% during the 9-yr study follow-up after adjusting for age, gender, race, and baseline eGFR.^[21] A meta-analysis by Tonelli et al.^[33] showed that individuals with mildly decreased kidney function (80 mL·min⁻¹·1.73 m⁻²) had an increased risk for all-cause mortality compared with individuals with a normal GFR (100 mL·min⁻¹·1.73 m⁻²). The analysis also showed a progressive risk for mortality as kidney function deteriorated. To reduce this excess burden of the disease, it is important to identify modifiable factors associated with kidney function at earlier stages of the disease to better direct our intervention efforts in an attempt to slow down the progression.

Lifestyle modifications, including physical activity, have proven to be effective in the prevention of chronic conditions such as diabetes and hypertension.^[2,26,36] The potential benefits of physical activity in preventing or delaying the development of diabetes and hypertension are significant considering the fact that these two conditions are the leading causes of CKD and ESRD.^[27,34] There have been numerous investigations into the relationship between physical activity and other risk factors for CKD; however, few studies have examined the relationship between physical activity and CKD or kidney function.^[4,10,18] Much of the existing literature focuses on patients on dialysis. Although exercise may be important for individuals on dialysis, CKD is associated with CVD, and those with CKD are more likely to die from CVD than they are to progress to ESRD.^[30] CKD in early stages is typically asymptomatic, and individuals are often unaware they have the condition.^[38] Analogous to hypertension, CKD can be a silent killer, which makes it important to understand the relationship between modifiable lifestyle factors, such as physical activity, and renal function in early stages of the disease.

The few studies that have examined the association between physical activity and kidney function at earlier stages of the disease used subjective measures such as questionnaires.^[9,12,37] Although questionnaires perform relatively well at capturing activities of moderate to vigorous intensity, they have questionable accuracy in assessing lower-intensity activities, which is the largest component of total physical activity in most people.^[14] To accurately assess total physical activity, which is composed of both light and moderate to vigorous physical activity, an objective measure is needed.^[17] Recent studies using objective measures of physical activity have increased our knowledge regarding the contribution of lower intensity physical activity in reducing metabolic. The Australian Diabetes, Obesity and Lifestyle Study (AusDiab) was a cross-sectional study of 11,247 men and women ≥25 yr. The study showed that time spent in activities of lower intensity and sedentary behavior are independently associated with 2-h fasting plasma glucose, waist circumference, and a clustered metabolic risk score (triglycerides, HDL cholesterol, systolic and diastolic blood pressure, fasting plasma glucose, and waist circumference).^[15,16] This study highlights the importance of examining lower-intensity activity and its relationship to disease outcomes.

To date, no previous study has looked at the association between an objective measure of physical activity and kidney function in a nationally representative sample. The purpose of this study was to determine the association between time spent at various intensity levels of physical activity (light, moderate to vigorous, and total) and sedentary behavior with kidney function in individuals with mild to moderate kidney disease in a large national data set.

For further information: http://www.medscape.com/viewarticle/746893?src=mp&spon=8

From BMC Musculoskeletal Disorders

Patient-Reported Outcome Questionnaires for Hip Arthroscopy

A Systematic Review of the Psychometric Evidence

Marsha Tijssen; Robert van Cingel; Nicky van Melick; Enrico de Visser

Authors and Disclosures

Posted: 08/09/2011; BMC Musculoskeletal Disorders. 2011;12 © 2011 BioMed Central, Ltd.

Abstract and Introduction

Abstract

Background Hip arthroscopies are often used in the treatment of intra-articular hip injuries. Patient-reported outcomes (PRO) are an important parameter in evaluating treatment. It is unclear which PRO questionnaires are specifically available for hip arthroscopy patients. The aim of this systematic review was to investigate which PRO questionnaires are valid and reliable in the evaluation of patients undergoing hip arthroscopy.
Methods A search was conducted in Pubmed, Medline, CINAHL, the Cochrane Library, Pedro, EMBASE and Web of Science from 1931 to October 2010. Studies assessing the quality of PRO questionnaires in the evaluation of patients undergoing hip arthroscopy were included. The quality of the questionnaires was evaluated by the psychometric properties of the outcome measures. The quality of the articles investigating the questionnaires was assessed by the COSMIN list.
Results Five articles identified three questionnaires; the Modified Harris Hip Score (MHHS), the Nonarthritic Hip Score (NAHS) and the Hip Outcome Score (HOS). The NAHS scored best on the content validity, whereas the HOS scored best on agreement, internal consistency, reliability and responsiveness. The quality of the articles describing the HOS scored highest. The NAHS is the best quality questionnaire. The articles describing the HOS are the best quality articles.
Conclusions This systematic review shows that there is no conclusive evidence for the use of a single patient-reported outcome questionnaire in the evaluation of patients undergoing hip arthroscopy. Based on available psychometric evidence we recommend using a combination of the NAHS and the HOS for patients undergoing hip arthroscopy.

For further information: http://www.medscape.com/viewarticle/747254?src=mp&spon=8

From Reuters Health Information

Different Autografts Prompt Similar Results in ACL Repair

NEW YORK (Reuters Health) Jul 29 - Eleven years after joining a randomized trial, most patients who had anterior cruciate ligament (ACL) reconstruction were doing well regardless of whether their grafts came from the hamstring or the patellar tendon.

Subjective outcomes, objective stability, and graft failure rates were similar, according to Dr. Matjaz Sajovic and colleagues at the Institute of Public Health of the Republic of Slovenia, Ljubljana.

But the patellar tendon group did have a higher prevalence of osteoarthritis at 11 years, "which is the reason why I prefer the use of hamstring tendon autograft in most of my patients," Dr. Sajovic told Reuters Health by email.

Dr. Sajovic performed all of the original ACL reconstructions.

As reported online June 28th in The American Journal of Sports Medicine, he and his colleagues followed 64 patients who had been assigned to receive either semitendinosus and gracilis autografts (STG) or patellar tendon (PT) autografts.

Follow-up for 11 years in 54 patients showed no significant differences in Lysholm score and Short Form-36 (reflecting health-related quality of life), KT-1000 arthrometer laxity testing, anterior knee pain, single-legged hop test, or other measures.

Grafts ruptured in two STG patients (6%) and four PT patients (12%).

Eighty-one percent of STG patients and 72% of PT patients were at their preinjury levels of activity

The PT group, however, had more positive pivot-shift tests (+1). And 84% in that group had evidence of osteoarthritis affecting the knee, compared to 63% in the STG group - for reasons the authors can't explain.

"The results of our study," the authors conclude, "substantiate the similarities found in previous reports that document that good or excellent results may be obtained in the majority of ACL reconstructions when using either PT or STG autografts."

For further information: http://www.medscape.com/viewarticle/747289?src=mp&spon=8

From Reuters Health Information

Major League Baseball Injuries Increasing

By Allison Bond

NEW YORK (Reuters Health) Jul 29 - Injuries in Major League Baseball players are on the rise, despite advances in conditioning methods and injury treatments, according to research published June 27th in the American Journal of Sports Medicine.

The new study -- only the second to examine injury patterns among major league baseball players -- used the number of players on the disabled list over a seven-year period to gauge the risk of injury.

"There is very little information about injuries and injury rates in major league baseball," said Dr. Kenneth L. Cameron, director of orthopedic research at Keller Army Hospital in West Point, New York and an author of the study. "We wanted to quantify the injuries in baseball at its highest level."

Analyzing the 2002 through 2008 seasons, his group found that player injury rates jumped by nearly 40% after 2005.

The increase may have been due in part to a crackdown on steroid use during that period, Dr. Cameron told Reuters Health, because performance-enhancing drugs can also speed recovery between games.

"When the drug policy changed, it may have affected injury rates," he said.

The nature of injuries did correspond fairly predictably to player positions, according to the research. Pitchers were more likely to injure their arms, wrists, elbows or shoulders, whereas fielders more often injured their legs and hips.

"These findings confirm what we could expect," said Dr. Douglas Comeau, a sports medicine physician and assistant professor at Boston University School of Medicine.

The most common injuries among fielders include hamstring and groin strains. Pitchers, on the other hand, tend to strain or tear ligaments in the elbow or rotator cuff, said Dr. Comeau.

In the study, pitchers got hurt most often, accounting for 62% of all disability days, versus 32% for fielders.

Overall, however, the frequency of injuries decreased every month from April to September -- the first and last months of the regular season.

The initial spike in injuries may be from a lack of conditioning prior to the first big games of the season, Dr. Comeau told Reuters Health. "Major league baseball players might not take spring training seriously, but then they need to play a whole game in a competitive situation."

Dr. Cameron and colleagues say their data may be used to develop an MLB injury database, they write, "as well as in the development and implementation of specific preseason training and in-season conditioning for injury prevention."

For further information: http://www.medscape.com/viewarticle/747296?src=mp&spon=8

From Reuters Health Information

Non-Head Injuries May Impact Thinking Skills: Study

By Genevra Pittman

NEW YORK (Reuters Health) Jul 28 - A blow to the head isn't the only injury that can make a football player a little slow and confused.

New research shows that a sprained ankle or strained knee might also affect how athletes perform on the Automated Neuropsychological Assessment Metrics (ANAM) test battery, a computer-based assessment of attention, memory, and reaction time generally used to manage concussions.

That means trainers and team doctors should rely on talking to athletes about concussion symptoms when they're trying to decide the seriousness of a head injury -- not just on the computer-based test, researchers said.

"With every athlete when we evaluate them on the sidelines...it's getting an idea of what's going on with that player, how they're feeling, what symptoms they may have," said Dr. Mark Halstead, an orthopedic surgeon at Washington University School of Medicine in St. Louis, who wasn't involved in the new study.

When it comes to concussions, "that's what we rely on most with these athletes," he told Reuters Health.

Doctors have become increasingly concerned in recent years about the consequences of concussions, especially in young athletes.

The head injuries can cause dizziness, memory problems, and trouble paying attention, which usually resolves in a few weeks. But when athletes try to come back from the blow too quickly, they risk developing the rare but dangerous "second impact syndrome," which happens when someone sustains a second head injury before the first is completely healed.

For the current study, researchers at the University of Toronto gave the 20-minute computerized battery of tests to 72 student-athletes, including football, hockey, and lacrosse players. Eighteen of those athletes had suffered a concussion in the past three days, and another 18 had been taken out recently by a muscle or tendon injury.

The other 36, used for comparison, were uninjured.

As expected, the head-injured athletes did worse than non-injured athletes, with slower reaction times and worse memory and attention. But those who had sustained a muscle or tendon injury also did worse than the healthy comparison group on some of the tests -- and their scores generally fell in between the scores of non-injured and head-injured athletes.

"It suggests that we need to take this testing with a grain of salt -- it isn't the end-all, be-all," Dr. Halstead said. "There may be some other factors that affect how someone performs on the test" besides how banged-up their brain is.

Those factors could include athletes' frustration with being injured, or other emotional responses to the injury, researchers led by Michael Hutchison said in a paper published online July 18 in the American Journal of Sports Medicine. Another possibility, they said, is that the pain and discomfort from tendon or muscle injuries might actually make athletes think less clearly.

"If you have someone that has had a head injury, you can't automatically assume everything you find (on the test) is head-injury related," Hutchison told Reuters Health. "It requires a little bit of interpretation."

Dr. Halstead said the results aren't a huge surprise, and that the computer test is only one tool doctors use to monitor how someone's doing after a head injury. "We can certainly manage a concussion fine without it," he said.

The findings show the importance of "using the whole picture, rather than just a test." But Dr. Halstead added that because the study was relatively small, further research will be needed to get the "big picture" of how non-head injuries may affect thinking skills -- and what that means for the tools doctors use to manage concussions.

For further information: http://www.medscape.com/viewarticle/747174?src=mp&spon=8

From Reuters Health Information

Platelet-Rich Plasma Has Little Effect on Rotator Cuff Healing

By Dave Levitan

NEW YORK (Reuters Health) Aug 03 - Giving platelet-rich plasma did not help patients recover from arthroscopic rotator cuff surgery in a South Korean study.

Platelet-rich plasma (PRP) is being used in various orthopedic procedures these days without a lot of evidence to support it, the study's authors say.

Led by Dr. Chis Hyunchul Jo of Seoul National University College of Medicine, they recruited 42 patients with full-thickness rotator cuff tears and let them choose on their own whether or not to have PRP applied in gel form during arthroscopic surgery.

As reported online July 7th in the American Journal of Sports Medicine, 19 patients chose the PRP therapy and 23 chose conventional therapy.

There were no significant differences between the PRP and conventional groups after 19.7 months of follow-up in terms of pain, range of motion, strength, and overall satisfaction measures.

Several functional scores were higher at three months postoperatively in the conventional therapy group, but there were no differences at the final follow-up.

Structural integrity, as determined by postoperative MRIs and retear rates, were also not significantly different between the two groups.

In an email to Reuters Health, Dr. Jo said the study could have been underpowered, and it suffered from other limitations as well.

"There were more large to massive tears in the PRP group," he said, and while the difference was not statistically significant, larger tears could delay the beginning of rehabilitation and mask the effects of treatment.

The lack of randomization also limits the interpretation of the results, Dr. Jo said.

The researcher team is not ready to give up on PRP yet, however. The paper concludes: "We do not regard our results sufficient to reach a conclusion regarding the effect of PRP on rotator cuff repair, and we suggest the need for further research."

For further information: http://www.medscape.com/viewarticle/747515?src=mp&spon=8

ARTHRITIS

ORTHOPEDICS August 2011;34(8):382.

Age-related Biological Characterization of Mesenchymal Progenitor Cells in Human Articular Cartilage

by Hong-Xing Chang, MD; Liu Yang, MD, PhD; Zhong Li, MD, PhD; Guangxing Chen, MD, PhD; Gang Dai, MD, PhD

DOI: 10.3928/01477447-20110627-06

Abstract

Adult articular cartilage has a low regeneration capacity due to lack of viable progenitor cells caused by limited blood supply to cartilage. However, recent studies have demonstrated the existence of chondroprogenitor cells in articular cartilage. A critical question is whether these mesenchymal progenitor cells are functionally viable for tissue renewal and cartilage repair to postpone cartilage degeneration.

This study was designed to compare the number and function of mesenchymal progenitor cells in articular cartilage collected from human fetuses, healthy adults (aged 28–45 years), and elderly adults (aged 60–75 years) and cultured in vitro. We detected multipotent mesenchymal progenitor cells, defined as CD105+/CD166+ cells, in human articular cartilage of all ages. However, mesenchymal progenitor cells accounted for 94.69%±2.31%, 4.85%±2.62%, and 6.33%±3.05% of cells in articular cartilage obtained from fetuses, adults, and elderly patients, respectively ( P<.001). Furthermore, fetal mesenchymal progenitor cells had the highest rates of proliferation measured by cell doubling times and chondrogenic differentiation as compared to those from adult and elderly patients. In contrast, alkaline phosphatase levels, which are indicative of osteogenic differentiation, did not show significant reduction with aging. However, spontaneous osteogenic differentiation was detected only in mesenchymal progenitor cells from elderly patients (with lower Markin scales). The lower chondrogenic and spontaneous osteogenic differentiation of mesenchymal progenitor cells derived from elderly patients may be associated with the development of primary osteoarthritis. These results suggest that measuring cartilage mesenchymal progenitor cells may not only identify underlying mechanisms but also offer new diagnostic and therapeutic potential for patients with osteoarthritis.

Drs Chang, Yang, Li, Chen, and Dai are from the Department of Joint Surgery, Southwest Hospital, Third Military Medical University, Chongqing, and Dr Chang is also from the Department of Orthopedics, Beijing Army General Hospital of PLA, Beijing, China.

Drs Chang, Yang, Li, Chen, and Dai have no relevant financial relationships to disclose. This study was supported by The National Natural Science Foundation of China (no. 30901576 and 30672200).

Correspondence should be addressed to: Gang Dai, MD, PhD, Department of Joint Surgery, Southwest Hospital, The Third Military Medical University, Chongqing 400038, China (daigang60@163.com).

Posted Online: August 08, 2011

Osteoarthritis results from an imbalance between damage to and repair of articular cartilage. Chondrocytes are thought to be terminal cells with limited capacity for proliferation, primarily because of poor blood circulation and a limited number of available stem cells. ¹ The articular cartilage is, therefore, often considered to be at high risk for age-related diseases. ^2,3 However, several recent studies have determined that chondrocytes from healthy primary osteoarthritis patients express markers specific for stem cells. ^4,5 These cells are called mesenchymal progenitor cells. Why, then, can’t the existing mesenchymal progenitor cells prevent or postpone cartilage degeneration? Previous studies have shown that aging stem cells from the bone marrow reduce the rate of generation and have a lower capacity of proliferation and differentiation. ^6,7 However, no report exists on the correlation between age and the rate of proliferation of cartilage mesenchymal progenitor cells.

We hypothesized that the ability of cartilage mesenchymal progenitor cells to proliferate and differentiate declines with age, leading to age-related decrease in chondrocyte numbers and function. This age-related decline in chondrocyte number and function directly contributes to the development of primary osteoarthritis. This study compared the in vitro functions of mesenchymal progenitor cells, defined as CD166+/CD105+ cells, isolated from cartilage and surgically removed from patients of different ages. The results may offer new insights into the pathogenesis of osteoarthritis and lead to the development of new preventive and therapeutic agents to improve the quality of life for patients with osteoarthritis.

Materials and Methods

Collection of Articular Cartilage

Fetal cartilage samples were obtained from fetuses aged 20 to 24 weeks who had died of congenital heart abnormalities. Samples of knee joint cartilage were taken from adult and elderly patients who had either died of other diseases or had undergone limb amputation. All cartilage was without visible joint disease (Table 1). Articular cartilage was dissected from the femoral condyle with perichondrium and subchondral bone excised and washed 3 times with phosphate buffered saline to remove blood and soft tissue on the surface. All samples were graded according to a modified Mankin scale (0–2). ⁸ Despite our efforts in choosing cartilage with minimal evidence of degeneration, cells collected for the study may have had age-associated degenerative changes in cartilage, which may have impacted the study. Samples were obtained after an informed consent form was signed by the patients and/or guardians. The study was approved by the hospital ethics committee.

: Cartilage Resource and Modified Mankin Scale8

Culture of Cartilage Cells

Articular cartilage cells were isolated as previously described. ⁹ Briefly, an articular cartilage was cut into 1-mm ³ pieces and incubated with phosphate buffered saline containing 0.1% trypsin, 0.1% hyaluronidase, and 0.2% type II collagenase at 37°C for 1 hour and 3 hours, respectively. Chondrocytes were harvested by centrifugation at 1000 rpm for 5 minutes and seeded into a cell culture flask at a plating density of 3×104 cells/cm ² in Dulbecco’s Modified Eagle Medium: Nutrient Mixture F-12 (DMEM/F-12) medium with 10% fetal bovine serum, 100 IU/mL penicillin, and 100 µg/mL streptomycin. The cell culture was maintained at 37°C with air that contained 5% (v/v) CO ₂. The medium was changed 72 hours after initial seeding, and chondrocytes continued to be cultured until 80% confluent. Cells were then detached with trypsin (0.25%) and reseeded at control densities for functional assays.

Detection of CD105+/CD166+ Cells in Chondrocytes From Articular Cartilage

Cells from the primary culture and after the second and fourth passages were detached with trypsin and resuspended to a final concentration of 105 cells/50 µl in phosphate buffered saline supplemented with 1% bovine serum albumin and incubated with phycoerythrin-conjugated anti-CD105 (monoclonal antibody SN6) and fluorescein isothiocyanate (FITC)-conjugated anti-CD166 antibodies for 45 minutes at 4°C. Cells stained with a mouse isotype IgG were used as negative control. After antibody binding, cells were washed 3 times with phosphate buffered saline containing 1% bovine serum albumin to remove unbound antibodies and fixed in 4% paraformaldehyde in phosphate buffered saline for 15 minutes at room temperature. They were subjected to fluorescence-activated cell sorting using a FACScan and analyzed with the CellQuest program (Becton Dickinson, San Jose, California).

For flow cytometry analysis, cells were first gated on the forward and side scatters to exclude debris and cell aggregates and then detected for specific antibody binding. The percentage of dual-positive cells was calculated after subtracting non-specific binding of mouse isotype IgG as described previously. ⁴

Isolation of Articular Cartilage Mesenchymal Progenitor Cells

An Anti-FITC MultiSort Kit and a miniMACS separation system were used to purify mesenchymal progenitor cells according to instructions from the manufacturer (Miltenyi Biotec, Bergisch Gladbach, Germany). Briefly, chondrocytes from the fourth passage in culture were harvested and suspended in phosphate buffered saline. They were incubated with an FITC-CD166 antibody (1:50 dilution) for 45 minutes in the dark at 4°C. The stained cells were washed twice and then resuspended in 80 µl of 1× phosphate buffered saline containing 1% bovine serum albumin and 0.1% NaN3. They were then incubated with 10 µl of an anti-FITC antibody coupled to magnetic beads for 30 minutes at 4°C. After washing, CD166+ cells were selected with a magnetic apparatus (Miltenyi Biotec). The selected CD166+ cells were further selected with CD105 micromagnetic beads (Miltenyi Biotec) to eventually obtain cells that are positive for CD166 and CD105. The sorted cells were cultured at a density of 5×104 cells/cm ².

Laser Scanning Confocal Fluorescence Microscopy

Magnetic bead-purified mesenchymal progenitor cells were either prepared as smears for histological examination or seeded on a 10×10-mm cover glass to be cultured in DMEM/F-12 culture medium for 3 days. Cells prepared in both techniques were fixed in 4% polyoxymethylene (30 minutes at room temperature). The fixed cells were first blocked with a buffer containing 5% fetal bovine serum and 2% bovine serum albumin for 30 minutes and then stained for phycoerythrin-conjugated CD105 (1:50 dilution) and FITC-conjugated CD166 (1:50 dilution) antibodies (phycoerythrin or FITC mouse IgG as controls) for 30 to 45 minutes at room temperature. The slides were counterstained with 4,6-diamidino-2-phenylindole (DAPI) to mark the nucleus. Fluorescently labeled cells were visualized and imaged under a confocal laser scanning microscope (Leica TCS SP5; Leica Microsystems, Wetzlar, Germany).

Cell Doubling Time

Cell doubling time (Td) was used to calculate the ability to proliferate. Mesenchymal progenitor cells in culture (second passage) were detached with trypsin (0.25%), suspended in DMEM/F-12 medium containing 10% fetal bovine serum, and plated into a 96-well plate (2×103 cells/well). Cells in 3 wells from each age group were processed in a standard MTT procedure ¹⁰ and examined every 24 hours (optical density, 570 nm). A growth curve was plotted from these optical density values to calculate mesenchymal progenitor cells-cell doubling time in each age group based on the following formula: Td=t*lg2/(lgNt-lgN0), where t is time in culture (hours), N0 is the seeding density of cells, and Nt is the cell density after t hours in culture.

Detection of Articular Cartilage Mesenchymal Progenitor Cells Differentiation

Chondrogenic Induction. Mesenchymal progenitor cells were cultured with chondrogenic medium (DMEM/F-12 supplemented with 1% ITS Liquid Media Supplement (Sigma-Aldrich Co, St Louis, Missouri), 1 mM sodium pyruvate, 37.5 g/mL ascorbate 2-phosphate, 10-8 M dexamethasone, and 10 ng/mL recombinant human transforming growth factor-ß1) for 2 weeks. ⁴ Control cells were cultured in DMEM/F-12 containing 10% fetal bovine serum.

After 2 weeks in culture, total RNA was isolated using TRIzol reagent (Tiangen Biotech Co Ltd, Beijing, China) and used for semiquantitative reverse transcription-polymerase chain reaction to detect the expression of type II collagen and aggrecan gene. Reverse transcription was first performed with 1 µg of total RNA from each sample using oligo(dT)18 primer and 200 units of SuperScript II RT (Life Technologies Inc, Gaithersburg, Maryland) for cDNA synthesis. DNA (in triplicate) was then amplified in 20 µL solution that contained 2 µl diluted template, 10 pmol primer pairs for type II collagen, and aggrecan and control glyceraldehyde 3-phosphate dehydrogenase (Table 2), respectively, and 10 µl Taq PCR Master Mix (TianGen Biotech Co Ltd). The amplification was induced first at 94°C for 5 minutes, followed by 30 cycles of 30 seconds at 94°C, 30 seconds at 57°C, and 30 seconds at 72°C. The reaction was completed by a final incubation at 72°C for 10 minutes. Gene expression was expressed as 2- ^??(Ct), where Ct is the cycle threshold, ?(Ct) is the Ct of the tested gene–Ct of glyceraldehyde 3-phosphate dehydrogenase, and ??(Ct) is the ?(Ct) of sample 1–?(Ct) of sample 2. ¹¹

: Primers Used for Real-time Polymerase Chain Reaction

Osteogenic Induction. Mesenchymal progenitor cells (1×104/cm ²) were first cultured in DMEM/F-12 medium for 24 hours and then switched to the osteogenic medium (50 µmol/L ascorbic acid, 10 µM ß-sodium glycerophosphate, and 0.1 µM dexamethasone) ¹² for 14 days. Control cells were cultured in DMEM/F-12 containing 10% fetal bovine serum. Intracellular alkaline phosphatase was measured with a commercial kit (LabAssay; Wako Pure Chemical Industries, Ltd, Osaka, Japan) at 405 nm and calculated as instructed by the manufacturer.

Statistical Analysis

All values are presented as mean±standard error of mean from repeated experiments. The quantitative data were analyzed using SPSS 13.0 software (SPSS, Inc, Chicago, Illinois). The Kruskal-Wallis test (non-parametric) was used to compare among multiple groups. The independent samples t test was used to assess difference between 2 groups of variables. A P value <.05 was considered statistically significant.

Results

CD105+/CD166+ Cells in Articular Cartilage

The percentage distributions of CD105+/CD166+, CD34+, and CD45+ cells purified from articular cartilage were similar to mesenchymal progenitor cells from the bone marrow (Figure 1), consistent with a previous report on mesenchymal progenitor cells. ⁴ In primary culture, articular cartilage from fetuses had the highest percentage of CD166+/CD105+ cells as compared to those from adult and elderly patients ( P<.001). There was no statistical difference in the counts of CD166+/CD105+ cells between articular cartilage obtained from adult and elderly patients. However, the percent of CD166+/CD105+ cells from adult and elderly patients significantly increased in culture, whereas those from fetal tissue (which counted for >90%) did not show significant changes (Table 3; Figure 2).

CD166-fluorescein isothiocyanate/CD105-phycoerythrin positive cells after immunomagnetic purification was identified by fluorescence-activated cell analysis (A). The same technology was also used to detect positivity for CD34 (0.50%) and CD45 (0.69%) (B). Laser scanning confocal fluorescence microscope detected mesenchymal progenitor cells after labeling with phycoerythrin-CD105 and fluorescein isothiocyanate-CD166 antibodies. DAPI (4',6-diamidino-2-phenylindole) was used to highlight the nucleus. Geometrical mean fluorescence was used to measure the antibody binding in fluorescence intently (X axis). Representative of 5 independent experiments (C).

: CD105+/CD166+ Mesenchymal Progenitor Cells in Articular Cartilage in Different Age Groups

Fluorescence-activated cell-sorting analysis of chondrocytes was presented on forward/side scatter plot for primary culture (A) and after 4 passages (B). The scatter plot was set on a linear scale to indicate particle size and was used to exclude cellular debris and aggregates. The CD166-fluorescein isothiocyanate/CD105-phycoerythrin staining was detected in the primary culture (C) and after 4 passages (D) in geometrical mean fluorescence.

Growth and Proliferation of Mesenchymal Progenitor Cells

The mesenchymal progenitor cells from fetal articular cartilage were short, spindle-shaped cells that grew rapidly, whereas most of the mesenchymal progenitor cells from articular cartilage of adult and elderly patients were longer spindle-shaped cells (Figure 3). The proliferation capacity was lower for mesenchymal progenitor cells from adult and elderly patients as compared to those from fetal samples, with the cell doubling times of 25.68±7.71 hours, 45.35±15.41 hours, and 55.69±16.52 hours for fetal-, adult-, and elderly-derived mesenchymal progenitor cells, respectively (Figure 4).

Morphological characterization of chondrocytes (A–C) and CD166+/CD105+ mesenchymal progenitor cells (D–F) from fetal, adult, and elderly patients, respectively, after 2 passages in culture.

Cell growth (1 passage in culture) was measured for mesenchymal progenitor cells from different age groups, indicating a higher grown rate for fetal mesenchymal progenitor cells (A). Cell doubling time was calculated for mesenchymal progenitor cells from different groups of patients, showing a lowest double time (Kruskal-Wallis test, P=.001) (B).

Chondrogenic Differentiation

mRNA for aggrecan and type II collagen were the highest in chondrogenic-induced fetal mesenchymal progenitor cells as compared to those from adult and elderly patients ( P<.05) (Figure 5). These results indicate that CD105+/CD166+ mesenchymal progenitor cells in the resting state differentiated into chondrocytes and upregulated the expression of aggrecan and type II collagen mRNA in the presence of growth factors.

mRNA for aggrecan and type II collagen was detected by real-time polymerase chain reaction with the unit of Y-axis as the relative expression level measured in quantitative polymerase chain reaction. The expression levels of aggrecan and type II collagen mRNA in fetal (group A), adult (group B), and elderly (group C) mesenchymal progenitor cells were 7.79 and 9.34 times the control, 5.82 and 7.02 times the control, and 2.24 and 2.93 times the control, respectively. The chondrogenic potential declines with age ( P<.01), and chondrogenic differentiation was also significantly reduced in aged cartilages.

Osteogenic Differentiation

Levels of alkaline phosphatase in mesenchymal progenitor cells from all age groups increased after osteogenic induction, indicating that cartilage mesenchymal progenitor cells from all ages maintained a normal osteogenic differentiation capacity. However, a 2-week induction resulted in a higher level of alkaline phosphatase in mesenchymal progenitor cells from adult patients as compared to those from fetal and elderly patients, but the difference did not reach statistical significance (Figure 6). Without induction, alkaline phosphatase of mesenchymal progenitor cells from elderly patients was significantly higher than that of the other 2 groups ( P=.007), suggesting that mesenchymal progenitor cells from elderly patients’ cartilage might have undergone spontaneous osteogenic differentiation.

The alkaline phosphatase (ALP) levels after osteogenic induction of mesenchymal progenitor cells show no significant difference among the 3 groups (P=.215), but show a significant difference among different patients without induction (controls).

Discussion

Chondrocytes are thought to be terminal cells with low capacity for reproduction or self-renewal. Blood circulation in articular cartilage is also poor, potentially resulting in limited supply of progenitor cells critical for tissue renewal. ^2,3 These factors contribute in part to a high risk for age-related disease in articular cartilage. However, recent studies have found that osteoarthritis chondrocyte express stem cell markers. ^4,5 A key question is whether these mesenchymal progenitor cells undergo functional changes that result in progressively reduced capacity for self-renewal and differentiation. We have provided experimental evidence that human cartilage of all ages contained CD105+/CD166+ mesenchymal progenitor cells, ^13,14 which are known to differentiate into mature cells of chondrogenic as well as adipogenic and osteogenic lineages. ^4,15 As expected, mesenchymal progenitor cells were found to have a higher quantity in fetal cartilage, consistent with a previous report. ⁴ Interestingly, mesenchymal progenitor cells in cartilage from elderly patients were similar in quantity to those from adult patients, indicating a minimal decline in mesenchymal progenitor cells between the 2 age groups.

Despite their universal presence, we found that fetal mesenchymal progenitor cells had a higher rate of proliferation and chondrogenic capacity as compared to aged cartilage (Figures 4, 5), similar to those found in bone marrow mesenchymal progenitor cells. ^6,16 This low rate of proliferation and slow induction is consistent with finding that aged cartilage had a lower percentage of mesenchymal progenitor cells that had a reduced chondrogenic capacity, as shown in Figure 5. A role of aging in mesenchymal stem cell differentiation has been debated. ^7,17 Our observations strongly suggest a mechanism that supports for age-related development of cartilage degeneration and osteoarthritis.

Cell growth (1 passage in culture) was measured for mesenchymal progenitor cells from different age groups, indicating a higher grown rate for fetal mesenchymal progenitor cells (A). Cell doubling time was calculated for mesenchymal progenitor cells from different groups of patients, showing a lowest double time (Kruskal-Wallis test, P=.001) (B).

mRNA for aggrecan and type II collagen was detected by real-time polymerase chain reaction with the unit of Y-axis as the relative expression level measured in quantitative polymerase chain reaction. The expression levels of aggrecan and type II collagen mRNA in fetal (group A), adult (group B), and elderly (group C) mesenchymal progenitor cells were 7.79 and 9.34 times the control, 5.82 and 7.02 times the control, and 2.24 and 2.93 times the control, respectively. The chondrogenic potential declines with age ( P<.01), and chondrogenic differentiation was also significantly reduced in aged cartilages.

Furthermore, spontaneous osteogenic differentiation in mesenchymal progenitor cells derived from elderly patients may be related to primary osteoarthritis. For example, mesenchymal progenitor cells cultured in a transforming growth factor-ß-containing chondrogenic medium display signs consistent with chondrocyte hypertrophy. ¹⁸ Osteoarthritis chondrocytes in culture show significant hypertrophy after transforming growth factor-ß induction, ¹⁹ which precedes cartilage apoptosis, vessel invasion, and calcification during cartilage development. Xiao et al ²⁰ compared the gene-expression pattern in the bone marrow-mesenchymal stem cells of geriatric (>2 years), osteoporotic and nonosteoporotic adult (7 months), and juvenile (7 weeks) rats and detected the highest high expression of osteoblast-related genes in geriatric rats. Together, these findings imply that mesenchymal progenitor cells from elderly animals could promote the development of osteoarthritis by differentiating into bone cells.

Although we have observed significant differences in mesenchymal progenitor cell function in patients from different age groups, the sample size for this study is too small to establish a linear relationship between age and mesenchymal progenitor cell functionalities, due to difficulties obtaining cartilage tissue. It may be more informative to study cartilage mesenchymal progenitor cells at epiphyses fusion age, when cartilage evolves from the growth phase to the stationary phase. This concern is partially addressed by examining fetal cartilage, which represents the growth phase, as well as adult and elderly cartilage, which represent the stationary and degenerative phases. Nevertheless, more studies on the subject are called for, including larger randomized controlled trials to study how mesenchymal progenitor cells associate with osteoarthritis development and to explore the potential of using mesenchymal progenitor cells as a therapeutic alternative to the standard care of patients with osteoarthritis.

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