I recently found a Wikipedia article (HERE) on a topic which made me really think about a critical element to what determines our overall height, which is our mental and emotional state.

We all know that genetics, sleep, nutrition, and exercise plays a role in determining our height, but what about our mental health? There are many claims going around the height increase and grow taller niche and one of them is that of using hypnosis. A lot of people state that for you to be taller, you have to really be positive and believe you WILL grow taller and increase your height. So the obvious question is. How much does our mental state affect our height?

From the article on Psychosocial Short Stature, it seems that the effects are really dramatic, at least when it some to how it can limit our grow if we are not in the healthiest of mental states.

From the wiki article…

Psychosocial short stature (PSS) or psychosocial dwarfism, sometimes called psychogenic or stress dwarfism, or Kaspar Hauser Syndrome,[3] is a growth disorder that is observed between the ages of 2 and 15, caused by extreme emotional deprivation or stress.

The symptoms include decreased growth hormone (GH) secretion, very short stature, weight that is inappropriate for the height, and immature skeletal age. This disease is a progressive one, and as long as the child is left in the stressing environment, his or her cognitive abilities continue to degenerate. Though rare in the population at large, it is common in feral children and in children kept in abusive, confined conditions for extended lengths of time. It can cause the body to completely stop growing but is generally considered to be temporary; regular growth will resume when the source of stress is removed.

Etiology

Children with PSS have extremely low levels of growth hormone. These children possibly have a problem with growth hormone inhibiting hormone (GHIH) or growth hormone releasing hormone (GHRH). The children could either be unresponsive to these hormones or too sensitive.

Children who have PSS exhibit signs of failure to thrive. Even though they appear to be receiving adequate nutrition, they do not grow and develop normally compared to other children of their age.

An environment of constant and extreme stress causes PSS. Stress releases hormones in the body such as epinephrine and norepinephrine, engaging what is known as the ‘fight or flight’ response. The heart speeds up and the body diverts resources away from processes that are not immediately important; in PSS, the production of growth hormone (GH) is thus affected. As well as lacking growth hormone, children with PSS exhibit gastrointestinal problems due to the large amounts of epinephrine and norepinephrine, resulting in their bodies lacking proper digestion of nutrients and further affecting development.

While the cure for PSS is questionable, some studies show that placing the child affected with the disease in a foster or group home increases growth rate and socialization skills.

Me: I asked a person I am working with on what she can find on the link between mental health and human growth and she pointed out two articles which I looked over. They are located HERE (resource 1) and HERE (resource 2).

From resource 1, HERE

Other endocrine interactions

Growth, reproductive function, and the thyroid axis are also influenced by stress system activation. In the acute setting of stress, glucocorticoids stimulate the growth hormone gene, leading to enhanced growth hormone secretion.⁵³ However, with more prolonged stress, growth hormone release is suppressed by CRH‐induced elevations in somatostatin levels.⁵⁴ This results in an inverse relationship between the diurnal concentrations of cortisol and growth hormone. Also, glucocorticoids directly inhibit growth hormone effects at target tissues by inhibiting insulin‐like growth factor‐1 (IGF‐1) and other growth factors.⁵⁵ The effects of stress on the growth axis may account for the delay in growth often seen in chronic disease and emotional deprivation in childhood.

CRH, β‐endorphin, and glucocorticoids inhibit GnRH secretion from the hypothalamus. Glucocorticoids also suppress pituitary gonadotrophin release and inhibit gonadal tissue.⁵⁶ Patients with illnesses associated with increased HPA‐axis activity, such as anorexia nervosa, hyperthyroidism, and malnutrition, may experience abnormalities of menstrual and reproductive function.^57–,⁵⁹

From resource 2, HERE

Me: I really coudn’t figure out the link to the study and how human height is effected. The conclusion is that behavior created from stress is from the Corticotropin-releasing hormone receptor, but don’t need the actual hormone. The Abstract below…

Stress-induced behaviors require the corticotropin-releasing hormone (CRH) receptor, but not CRH

• Stacie C. Weninger*†, Adrian J. Dunn‡, Louis J. Muglia†§, Pieter Dikkes¶, Klaus A. Miczek‖, Artur H. Swiergiel‡, Craig W. Berridge**, and Joseph A. Majzoub†‡‡

+

Author Affiliations

• ^*Program in Neuroscience, Howard Hughes Medical Institute, and ^†Division of Endocrinology and ^¶Department of Neurology, Children’s Hospital, and Harvard Medical School, Boston, MA 02115; ^‡Department of Pharmacology, Louisiana State University Medical Center, Shreveport, LA 71130; ^‖Department of Psychology, Tufts University, Medford, MA 02155; and ^**Department of Psychology, University of Wisconsin, Madison, WI 53706

• Edited by Bruce S. McEwen, The Rockefeller University, New York, NY, and approved April 30, 1999 (received for review October 6, 1998)

Next Section

Abstract

Corticotropin-releasing hormone (CRH) is a central regulator of the hormonal stress response, causing stimulation of corticotropin and glucocorticoid secretion. CRH is also widely believed to mediate stress-induced behaviors, implying a broader, integrative role for the hormone in the psychological stress response. Mice lacking the CRH gene exhibit normal stress-induced behavior that is specifically blocked by a CRH type 1 receptor antagonist. The other known mammalian ligand for CRH receptors is urocortin. Normal and CRH-deficient mice have an identical distribution of urocortin mRNA, which is confined to the region of the Edinger–Westphal nucleus, and is absent from regions known to mediate stress-related behaviors. Since the Edinger–Westphal nucleus is not known to project to any brain regions believed to play a role in anxiety-like behavior, an entirely different pathway must be postulated for urocortin in the Edinger–Westphal nucleus to mediate these behaviors in CRH-deficient mice. Alternatively, an unidentified CRH-like molecule other than CRH or urocortin, acting through the CRH receptors in brain regions believed to mediate stress-induced behaviors, may mediate the behavioral response to stress, either alone or in concert with CRH.

Stress, defined as the response of the body to any threatening demand (1), can be broadly separated into physiological responses, including the stimulation of adrenal glucocorticoid secretion, and behavioral responses, including anxiety and fearful behavior. Alterations in the stress-response system are believed to underlie many anxiety-related disorders (2–5). Corticotropin-releasing hormone (CRH) has been implicated in both physiological and behavioral stress responses. CRH was identified by its ability to stimulate adrenocorticotropic hormone (ACTH) secretion from anterior pituitary corticotrophs, thus activating the hypothalamic–pituitary–adrenal (HPA) axis (6). In addition, infusion of CRH into the brain was found to cause stress-like behaviors (7), suggesting that CRH integrates physiological and behavioral activities into a generalized stress response. Subsequently, many other pharmacological studies have implicated CRH in the behavioral response to stressors. These studies confirmed that intracerebral infusion of CRH induces stress-like behaviors and additionally that intracerebral infusions of CRH antagonists blunt the behavioral response to a stressor (8–10). Furthermore, transgenic mice that overexpress CRH exhibit increased anxiety-like behavior (11).

The role of the CRH receptor in the behavioral stress response has been further evaluated. The two known CRH receptors, type 1 and type 2 (12), both consist of a 7-transmembrane helix functionally coupled to adenylate cyclase via G_s. For the most part, the anatomical distributions of the two CRH receptors are distinct, with the type 1 receptor expressed in the central nervous system in regions including neo-, olfactory, and hippocampal cortices, subcortical limbic structures in the septal region and amygdala, certain relay nuclei in the brainstem, and the cerebellum (13). One splice variant of the type 2 receptor is expressed in the periphery, whereas another splice variant is expressed in specific subcortical structures, the lateral septal nuclei, and the hypothalamus (14, 15). Two reports of mice lacking the CRH type 1 receptor have confirmed a role for this receptor in anxiety-related behavior (16, 17). CRH type 1 receptor-deficient mice display decreased anxiety-like behavior in the dark–light emergence task and the elevated-plus maze, both behavioral paradigms thought to measure anxiety in rodents. Both studies conclude that CRH mediates the behavioral responses to stressors by means of the CRH type 1 receptor (16, 17). More recently, a CRH type 1 receptor antagonist has been found to block both the acquisition and expression of stress-induced behaviors in rats (18).

Thus, the CRH type 1 receptor clearly mediates behavioral responses to stress, with the evidence to date implicating CRH as its most likely ligand (9, 10, 19). The other known mammalian ligand for CRH receptors is urocortin (20). Urocortin, acting either alone or in concert with CRH, is a potential mediator of in vivo stress responses, because in the studies cited above, infused or transgenically overexpressed CRH could act at receptor sites normally occupied by urocortin, and CRH antagonists could block urocortin actions as well as those of CRH (21). Therefore, we examined the role of CRH in this pathway by characterizing the behavioral responses to stressors of CRH-deficient (knockout, KO) mice (22) and the effects of CRH antagonists on these responses. We also examined the expression of urocortin mRNA in wild-type (WT) and CRH KO mouse brain to assess whether its distribution provides insight into its potential role in mediating stress-induced behaviors.

Me: What I can say is that stress does cause the human brain to release certain hormones that seems to inhibit the human longitudinal growth process. Here is my theory, from using evolutionary biology. The human body can be viewed as a system of work done that needs energy input. For the body/system to become bigger, it needs a lot more energy than before to even get it to grow to that size. 

So, while you are still young, stay happy and dont get too stress out. There is really no good point and you won’t be inhibiting your growth process.

If we view stress, as a form of resistance or internal conflict, we can use the analogy froom physics that stress is like friction, which is lost energy. If we are losing the energy, we don’t have the energy to go through our growth spurts. So stress is taking away the energy our young bodys/minds need to make that quantum energy leap. 

From the researcher, she states…

“cortisol is released during stress, and cortisol inhibits growth,….Yes, being in a happier less stressful situation, a person raising their chances of growing.”

Me: I wanted to make this post be about using surgery to correct for spinal deformities. In effect, the rod implanted into the vertebrate to stop the curvature of the spine and relieve much pain will also increase height.

I found this article HEY Clinic For Spine & Surgery at Raleigh, NC where a spine surgeon talks about using a new 4-rod implant to make a patient who already had implants in her back stronger and more durable since her current rods have broken apart..

The Hey Clinic for Scoliosis and Spine Surgery

This is where Dr. Hey shares his thoughts regarding recent patients he has cared for, and shares photos and case summaries to benefit patients, their families and friends, other physicians, medical students, residents, and fellows. Dr. Hey works at the Hey Clinic for Scoliosis and Spine Surgery, Raleigh, NC USA http://www.heyclinic.com.

FRIDAY, SEPTEMBER 14, 2007

Hey Clinic Sep 13 2007 AM Surgery: New “QUAD ROD” technique for stabilizing thoracic kyphotic fracture with history of rod breakage.

Yesterday morning, September 13th 2007, we helped a Nancy, a 75 yo woman who had a T12 severe wedge fracture above a previous fusion which caused myelopathy and severe pain. This was fixed using a laminectomy, and extension instrumentation and fusion. Postoperatively, she had trouble with rod breakages at that high load area around T12, with 2 rod construct, and then 3 rod construct. She how presented with recurrent rod breakage at that same T12 area, breaking both rods, and breaking conectors for the triple rod, resulting in recurrent kyphosis and pain. Given the amount of wedging at T12, we had discussed possibility of fixing this problem with an anterior/posterior procedure, removing body of T12 and replacing the vertebra with a strut expandable cage with graft, then instrumenting posteriorly. However, that is a very big operation, which involves having to take down diaphragm, etc., and Nancy was not interested in that at all. So, for the past couple of months, I have been working on several different engineering designs to make a stronger construct across this junction, “playing” with my set of Synthes “erector set” tools, and trying to come up with a new design which would be at least ten times stronger than the last one, but also be low profile enough that it would not cause problems with soft tissue prominence.

What seemed to be the best solution is a new surgical technique that I call the “Quad Rod”. Instead of just having 2 rods going across the area of the fracture, there are actually four rods, all closely coupled to each other with very strong rod to rod connecters. The combination of this second rod on each side closely coupled to the main rod means that there is major improvement in bending and twisting strength and rigidity, while still keeping a low profile instrumentation, while leaving the lateral “gutter” over the transverse processes wide open for BMP and bone graft.

Over the past couple months, I have done a bunch of different permutations of the “Quad Rod” on the spine model, and also experimented with other combinations including a 6 rod construct, which proved to be very strong, but very bulky. I also got a chance to try a small version of the Quad Rod unilaterally on a surgery a couple weeks ago which went well, and helped me think through some of the complexities of the rod insertion.

For my resident and fellow and younger spine surgeon readers, let me go over a few basic points.

1. Rods and/or screws are not meant to be able to withstand a lifetime of loading for most normal-sized people: they are meant to hold things in position hopefully long enough for the bone to heal, forming a fusion along the spine. This fusion can be thought of as “cement” that very slowly “hardens” around the metal superstructure, forming a very solid construct. However, the bone can take up to a year or even more to completely heal, especially in older patients who have weaker bone that does not make bone as quickly. If the bone does not heal, it is possible that the rods could break, or that the screws could loosen in the bone, and start to “toggle”. This could cause pain and deformity.

2. Whenever you face a revision instrumentation case, you always want to be thinking about making your new construct much stronger than the old one, while trying to get the bone to heal quicker using biological enhancers like bone morphogenic protein (BMP) and local or autologous bone graft. My military analogy here is this: If you get beaten in battle with 20 guys, don’t go back the next time with 20 or 21 guys —-> bring 100+ guys the next time, maybe with fire power from aircraft and a tank or two!

In this particular case, my first revision instrumentation used a triple rod technique with BMP, which had been my rock-solid revision instrumentation technique for broken rods, which had worked without fail for past 3 years or so without fail… Until Nancy broke the Triple Rod a couple months ago! So this time I am calling out the Marines, Air Force, Army, Navy and the Six Million Dollar Man! Here are the things I planned to do, and did differently with this revision:

1. Replace the screws above and below the unstable fracture with new screws, at least 1mm in diameter larger than the last ones. Putting in new screws here helps since the old ones had a lot of “cycles of load” on them and could break. 1mm larger diameter for each of these screws also makes the screw much stronger — the strength of the screw goes up as the 4th power of the diameter! (That’s like way bigger than double, or square or triple). So, for example, if you were to double the diameter of the screw, the strength would go up 16 time!

2. Put pedicle screws into the body of T12, where the fracture occurred. While I could not do this originally when the fracture occurred, since the body was “smashed”, now that it has been a year or more since her original fracture, I had hoped that the bone would have healed. Much to my delight today, I found that the bone had healed. It was still very tricky to get the screws placed, given previous laminectomy and scar, etc, but thankfully I got in 2 very solid screws at T12. This extra vertebral body support directly adjacent to where the fracture was unstable helped tremendously to improve the strength of the overall construct.

3. Stronger Rods. Thankfully, Synthes just recently released a new Titanium Alloy rod which is much stronger than the old rod, but is the same 7mm diameter. One of the fun things that occurred in this particular case was my interactions not only with Nancy, but with Nancy’s husband, Bob, who has a phenomenal background in metallurgy and engineering. One of Bob’s initial suggestions for this last revision was to use larger diameter rods, which from an engineering standpoint makes a lot of sense, but from a biologic standpoint doesn’t work as well, since the rods would become prominent. It would also require all of the screws and other connectors to be re-machined, and then retested. The best we could do was to get a stronger rod, but in the same diameter by changing the titanium alloy.

4. “HEY QUAD ROD”. This is one strong construct. The close coupling of 2 rods almost right next to each other with three encasing titanium block sleeves, which are then locked to each other with set screws makes for an incredible “I-Beam” that resists bending and twisting greatly. We recently received a grant, and are working with Professor Mazzolini and others at NC State Department of Mechanical Engineering to study the strength of some of these new constructs using computer models and lab testing. This “Quad Rod” is tough to put together, but once together is very low profile, and very strong. I have a bunch of tips that make it work very well. I used the new Quad Rod with the Synthes USS Titanium system, which has a special cap and nut locking system that locks the rod to the screw. One of the troubles you may have trying to do the Quad Rod with another system is that the screw diameter may be too large to allow the second joining rod to fit with the rod to rod connector. The rod to rod connector is something that other systems may have, which is used usually for doing extension instrumentations, where one rod is added onto another rod. In this case, I actually used a total of 5 rod to rod connectors on each side, and 10 altogether: three are used around the apex of the fracture, with the middle one right over the T12-L1 toggle point, and then one above at T11-T12 interspace, and L1-L2 interspace just below the apex of the “toggle”. The other 2 rod to rod connectors are placed at the top and bottom of the whole section of rod I replaced, which were down at L2 and up at T6 on each side. It turns out that the rod to rod connectors are almost the perfect length to “fill” the interspace between each of the pedicle screws, creating an additional thick “sheath” around the rod for extra bending resistance. The closely coupled rod next to the main rod provides additional bending and twisting support as a very tightly joined and/or cross-linked rod.

5. Additional cross-link between L and R rods just above the Quad Rod. There was too much fusion mass below the Quad Rod to put another cross-link, plus the large fusion mass below worked like a “cross link”.

6. Very aggressive cleanout of the pseudarthrosis at T12-L1, and the lateral “gutter” on either side, out over some of medial rib on both sides. This decoricated old fusion mass made a great new organic “bed” for two long strips of Medtronic Bone Morphogenic Protein (BMP) soaked sponges, followed by a layer of local bone graft, followed by a thick layer of allograft finely morselized chips.

Overall, the surgery went very well, taking right around 4 hours total.
I was able to accomplish all six of the steps above, although there were some real “fiddle factors” that needed to be dealt with. Here are a few pearls:

1. Your replacement long rod needs to be just the right length to connect between the very top and bottom connectors, but not significantly longer. When you go to insert the final construct, you need to slide it in one end, and then reduce it into the screw channels above, and then slide it up and into the rod to rod connectors at the very top.
2. Keeping those top and bottom connectors a little bit loose so they can rotate helps to get the new rod into the hole, and then rotated down into position and into the screw slots.
3. Measure your “partner rod”, the second rod on each side to bridge across 3 interspaces around the max stress point, with middle rod to rod connector over where the rods broke the last time.
4. Bend the rod in that area for both rods as little as possible, to avoid weakening the rod, and also to allow the rod to rod connectors to slide easily.
5. Attach the “partner rod” before inserting the overall construct, and put it medial to the main rod, with pedicle screw openings also facing medial. You will not be able to slide in partner rod when the first rod is in place. Placing it medial leaves the lateral gutter totally free for BMP and bone graft.
6. Mark the main rod with a pen where the 3 rod to rod connectors need to go to fall between the pedicle screws.
7. Put the middle of the 3 connectors on the partner and main replacement rod first, then slide the other two connectors over either end. Much easier than trying to guide the smaller partner rod through 3 connectors.
8. Insert the longer replacement rod at one end of the distal connectors first, as mentioned above, and then slide it into the upper connector, taking advantage of the rod to rod connector’s ability to rotate 90 degrees, and then down into position next to the screws.
9. Use the “Pursuader” to push the rod into the pedicle screws — it even works across the two rod construct! Tighten your set screws a little bit before you “pursuade” the double-rod construct into the screw slot.
10. As you are tapping the cap down over the main rod, there is very little space between the 2 rods — back off the compressor on the Pursuader a little bit, and the narrow “skirt” of the cap will go right between the 2 rods, and lock into position. Yahoo!
11. Torque down your set screws before tightening your nuts on top of the caps, for maximum strength.
12. Do thorough cleanout of lateral gutter and takedown of pseudarthrosis.
13. Strongly consider prior to placing rods putting additional screw points of fixation around the area of the “toggle” as I did, by putting T12 screws into old fractured vertebra. The more points of fixation you have, the better the load is shared to the spine, with less bending moment between screws.
14. Cut the BMP sponges into 2 longitudinal strips, and lay them down as a floor to the “gulley” over the transverse processes, ribs, and old fusion mass.
15. Undercut the paraspinal muscle flaps to allow for tension-free fascial closure at the end of surgery.

We will be studying this and other constructs in the future.

I encourage you to spend a lot of time “playing” with this and other constructs, using the plastic spine models with points of fixation, but also experimenting with other potential constructs as a pure erector set, to understand the possibilities. I see some possible very interesting constructs by joining 2 rod to rod connectors serially together, forming a double hinge. This allows you to have a “Quad Rod” where the rods are further apart, thereby potentially creating a better “I Beam” , by increasing the distance between the 2 beams. However, there are prominence problems, and the need for 2 connectors connected by a short intermediate rod I think is not half as strong as having 2 rods locked side by side. Perhaps a wider rod to rod connector would be a possibility, to allow the second rod to have more flexibility as to where it lies, and to potentially increasing the distance between the rods to increase strength.

Below are some pictures of our performance of our first real “Hey Quad Rod”.

I am happy to say that we got an excellent correction of her deformity, and we still have load sharing on the middle column anteriorly. However there is a big “divit” in the anterior portion of T12, which would have been nice to “fill in” with an anterior strut, but too invasive for Nancy. Our posterior Quad Rod tension band, combined with our “quick dry” BMP/bone graft combination, combined with even stricter postoperative patient restrictions for bending and lifting should hopefully lead to a long-term fix for Nancy.

This evening she was looking great in ICU, ready to get up and be probably 3-4 inches taller.

I hope these notes and pictures are helpful to my fellow surgeons out there. Please do not hesitate to call or email me if you have further questions or thoughts.

My email is “hey” at heyclinic.com, and my phone is available through website http://www.heyclinic.com.

Lloyd A. Hey, MD MS

Hey Clinic for Scoliosis and Spine Surgery

Raleigh, NC USA

http://www.HeyClinic.com

This article was sort of in the middle of nowhere and when I found it, I thought it was written by Tyler.  However, Tyler did comment to it. I wanted to continue this discussion further to get some more thought going on this topic.

Of all the articles written about height increase, this article was one of the most advanced and scientifically valid I have seen.

The areticle is found HERE.

Height Increase-Do you realize how close we are?

PLEASE NOTE: The validity of this article has been questioned. We do not have the resources to verify its claims, so please read the article with this in mind. We do not endorse or promote the views expressed by this guest post writer.

It’s frustrating that we are so close to finding a way for people to increase height, but we’re held up because scientists have to get funding and they’re not doing the right studies. Do you realize that their are no studies on height increase with mesenchymal stem cells? I mean come on harvest some red bone marrow from the trabecular bone and inject it into the hyaline cartilage growth plate line. Do you realize that hyaline cartilage composes the resting zone of the growth plate and since it has already excreted extracellular matrix, it does not undergo apoptosis like the rest of the chondrocytes in the growth plate?

Mesenchymal stem cells by definition are capable of inducing chondrogenesis. Scientists have found that a stem cell-like cancer cell was able to re-create a growth plate in hyaline cartilage. We are so close and yet we have to wait for the scientific method to go through it’s big, ugly steps.

Do you realize also that dynamic compressive loading of chondrocytes has been shown to alter gene expression in the cartilage? What would happen if this gene expression induced chondrogenesis in the hyaline cartilage in the growth plate line? Hmm, height increase by mechanotransduction.

It is so frustrating to see people get caught up in trying to grow taller with HGH or IGF-1 when their are so many other answers available. If IGF-1 works then it works by increasing Mesenchymal Stem Cell proliferation and Lithium also increases mesenchymal stem cell proliferation but it has the advantage of being legal (Me: I don’t believe this part is true. I needed Lithium once and I had to go to a doctor for this). The reason that gigantism works the way it does is by an alteration in the bodies homeostasis. It’s not just increased levels of HGH, the tumor in the pituitary gland(or whatever it is that alters the bodies homeostasis) works by altering the bodies negative feedback mechanisms to high levels of HGH. Robert Wadlow never stopped growing. You know why? Because his elevated levels of IGF-1 increased stem cell proliferation to the point where it never stopped.

The growth plates do not fuse. The hyaline cartilage is still there, it just becomes inactive. – (Me: This is the part which is controversial)

We are so close to finding a way to increase height but we’re too caught up on HGH and not caught up enough on mutagenics and stem cells. Do your part to help change that.

{ 2 comments… read them below or add one }

anonymous

There are no studies on height increase with mesenchymal stem cell because it is not worth considering. You can’t increase height with mesenchymal stem cell after puberty or prepuberty.

Even though there’s a little interrelationship between mesenchymal stem cell and epiphyseal plates, that doesn’t mean you can increase height with mesenchymal stem cell.

You need to do some more research.

Tyler Davis

There are some inaccuracies in this article that I the author want to correct:

The hyaline cartilage does disappear after endochondral ossification but as a separate process then chondrocytes differentiating into bone cells. However, stem cells don’t need hyaline cartilage to differentiate into chondrocytes.

Also, Lithium Supplementation is not legal without a prescription. However, having the Lithium ion in your body is legal.

If you have information about why mesenchymal stem cells can’t cause height growth I would like to hear them.

Me: I have looked at the same research and it appears the growth plate line does completely disappear over time. Stem cells have shown to be able to differentiate. Even though much of the original writer’s points are flawed, I think he/she does raise a really good point. I do believe also that we have the science and theory down well enough to develop some form of surgical process that allows us to implant a growth plate/stem cells/ hyaline cartilage  implant and make it work.

For anyone who is willing to separate their legs just temporarily, we can add a 3-4 mm thick plate with the right layers inside and allow endochondral ossification to do the rest of the work. All that is needed is to be able to inject stem cells into the plate or get the stem cells inside the bone to differentiate into the right types of chondrocytes. 

If anyone can show me why that can’t work, they have to go into some deep explanation since everything I have learned and read so far shows that it is already possible to get people to increase in height through stem cell implantations.

Released: 7/17/2012 1:30 PM EDT 

Source: Johns Hopkins Medicine

Newswise — Johns Hopkins tissue engineers have used tiny, artificial fiber scaffolds thousands of times smaller than a human hair to help coax stem cells into developing into cartilage, the shock-absorbing lining of elbows and knees that often wears thin from injury or age. Reporting online June 4 in the Proceedings of the National Academy of Sciences, investigators produce an important component of cartilage in both laboratory and animal models. While the findings are still years away from use in people, the researchers say the results hold promise for devising new techniques to help the millions who endure joint pain.

“Joint pain affects the quality of life of millions of people. Rather than just patching the problem with short-term fixes, like surgical procedures such as microfracture, we’re building a temporary template that mimics the cartilage cell’s natural environment, and taking advantage of nature’s signals to biologically repair cartilage damage,” says Jennifer Elisseeff, Ph.D., Jules Stein Professor of Ophthalmology and director of the Translational Tissue Engineering Center at the Johns Hopkins University School of Medicine.

Unlike skin, cartilage can’t repair itself when damaged. For the last decade, Elisseeff’s team has been trying to better understand the development and growth of cartilage cells called chondrocytes, while also trying to build scaffolding that mimics the cartilage cell environment and generates new cartilage tissue. This environment is a 3-dimensional mix of protein fibers and gel that provides support to connective tissue throughout the body, as well as physical and biological cues for cells to grow and differentiate.

In the laboratory, the researchers created a nanofiber-based network using a process called electrospinning, which entails shooting a polymer stream onto a charged platform, and added chondroitin sulfate—a compound commonly found in many joint supplements—to serve as a growth trigger. After characterizing the fibers, they made a number of different scaffolds from either spun polymer or spun polymer plus chondroitin. They then used goat bone marrow-derived stem cells (a widely used model) and seeded them in various scaffolds to see how stem cells responded to the material.

Elisseeff and her team watched the cells grow and found that compared to cells growing without scaffold, these cells developed into more voluminous, cartilage-like tissue. “The nanofibers provided a platform where a larger volume of tissue could be produced,” says Elisseeff, adding that 3-dimensional nanofiber scaffolds were more useful than the more common nanofiber sheets for studying cartilage defects in humans.

The investigators then tested their system in an animal model. They implanted the nanofiber scaffolds into damaged cartilage in the knees of rats, and compared the results to damaged cartilage in knees left alone.

They found that the use of the nanofiber scaffolds improved tissue development and repair as measured by the production of collagen, a component of cartilage. The nanofiber scaffolds resulted in greater production of a more durable type of collagen, which is usually lacking in surgically repaired cartilage tissue. In rats, for example, they found that the limbs with damaged cartilage treated with nanofiber scaffolds generated a higher percentage of the more durable collagen (type 2) than those damaged areas that were left untreated.

“Whereas scaffolds are generally pretty good at regenerating cartilage protein components in cartilage repair, there is often a lot of scar tissue-related type 1 collagen produced, which isn’t as strong,” says Elisseeff. “We found that our system generated more type 2 collagen, which ensures that cartilage lasts longer.”

“Creating a nanofiber network that enables us to more equally distribute cells and more closely mirror the actual cartilage extracellular environment are important advances in our work and in the field. These results are very promising,” she says.

Other authors included Jeannine M. Coburn, Matthew Gibson, Sean Monagle and Zachary Patterson, all from Johns Hopkins University.

The research was supported by grants R01 EB05517, F31 AG033999 and F30 AG034807 from the National Institutes of Health.

Found from the website Arthritis Research & Therapy

[Note: Just read the very first beginning part where everything is summarized in the abstract, introduction, results, and conclusion. This article is very long ]

Me: So the study was mainly done to see how to grow articular cartilage, not the type found in the growth plates exactly. 

Properties and usefulness of aggregates of synovial mesenchymal stem cells as a source for cartilage regeneration

Shiro Suzuki1, Takeshi Muneta1^,2, Kunikazu Tsuji2, Shizuko Ichinose3, Hatsune Makino4, Akihiro Umezawa4 and Ichiro Sekiya5^*

• *
  Corresponding author: Ichiro Sekiya sekiya.orj@tmd.ac.jp

Author Affiliations

¹Section of Orthopedic Surgery, Graduate School, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8519, Japan

² Global Center of Excellence Program, International Research Center for Molecular Science in Tooth and Bone Diseases, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8519, Japan

³ Instrumental Analysis Research Center, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8519, Japan

⁴ Department of Reproductive Biology, National Research Institute for Child Health and Development, 2-10-1 Okura, Setagaya-ku, Tokyo 157-8535, Japan

⁵ Section of Cartilage Regeneration, Graduate School, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8519, Japan

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Arthritis Research & Therapy 2012, 14:R136 doi:10.1186/ar3869

The electronic version of this article is the complete one and can be found online at: http://arthritis-research.com/content/14/3/R136

© 2012 Suzuki et al.; licensee BioMed Central Ltd.

This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

Introduction

Transplantation of mesenchymal stem cells (MSCs) derived from synovium is a promising therapy for cartilage regeneration. For clinical application, improvement of handling operation, enhancement of chondrogenic potential, and increase of MSCs adhesion efficiency are needed to achieve a more successful cartilage regeneration with a limited number of MSCs without scaffold. The use of aggregated MSCs may be one of the solutions. Here, we investigated the handling, properties and effectiveness of aggregated MSCs for cartilage regeneration.

Methods

Human and rabbit synovial MSCs were aggregated using the hanging drop technique. The gene expression changes after aggregation of synovial MSCs were analyzed by microarray and real time RT-PCR analyses. In vitro and in vivo chondrogenic potential of aggregates of synovial MSCs was examined.

Results

Aggregates of MSCs cultured for three days became visible, approximately 1 mm in diameter and solid and durable by manipulation; most of the cells were viable. Microarray analysis revealed up-regulation of chondrogenesis-related, anti-inflammatory and anti-apoptotic genes in aggregates of MSCs. In vitro studies showed higher amounts of cartilage matrix synthesis in pellets derived from aggregates of MSCs compared to pellets derived from MSCs cultured in a monolayer. In in vivo studies in rabbits, aggregates of MSCs could adhere promptly on the osteochondral defects by surface tension, and stay without any loss. Transplantation of aggregates of MSCs at relatively low density achieved successful cartilage regeneration. Contrary to our expectation, transplantation of aggregates of MSCs at high density failed to regenerate cartilage due to cell death and nutrient deprivation of aggregates of MSCs.

Conclusions

Aggregated synovial MSCs were a useful source for cartilage regeneration considering such factors as easy preparation, higher chondrogenic potential and efficient attachment.

Introduction

Synovial mesenchymal stem cells (MSCs) are an attractive cell source for cartilage regeneration because of their high expansion and chondrogenic potentials [1-5]. We previously reported that more than 60% of synovial mesenchymal stem cells placed on osteochondral defects adhered to the defect within 10 minutes and promoted cartilage regeneration [6,7]. With this local adherent technique, we can transplant synovial MSCs without scaffold. One of the disadvantages in this method is that the cell component in the suspension is invisible to the naked eye.

One of the solutions for this problem is to make aggregates of synovial MSCs [8-10]. This could enable MSCs not only to be visible but also to be heavier. Consequently, aggregates of MSCs will sink faster in the suspension medium than dispersed MSCs. The use of aggregates of MSCs may help to avoid loss of MSCs from targeted cartilage defects and improve the procedures of transplantation of synovial MSCs. However, there are still concerns; properties of synovial MSCs will be altered when synovial MSCs are aggregated. We do not know whether aggregates of MSCs adhere on the cartilage defect as we expect it will, and the proper number of aggregates is unclear.

In this study, properties of aggregates of human synovial MSCs were analyzed from the standpoints of morphology, gene profile and in vitro chondrogenic potential. Also, the effect of transplantation of aggregates of synovial MSCs was investigated in a rabbit cartilage defect model in terms of aggregate number, cell behavior and influential factors in the in vivo chondrogenesis of aggregates of synovial MSCs. Finally, we demonstrated the usefulness of aggregates of synovial MSCs as a source for cartilage regeneration therapy.

Materials and methods

Isolation and culture of human synovial MSCs

This study was approved by an institutional review board of Tokyo Medical and Dental University (No.1030), and informed consent was obtained from all subjects. Human synovium was harvested from donors during anterior cruciate ligament reconstruction surgery for ligament injury and digested in a 3 mg/ml collagenase D solution (Roche Diagnostics, Mannheim, Germany) in α-minimal essential medium (αMEM) (Invitrogen, Carlsbad, CA, USA) at 37°C. After three hours, digested cells were filtered through a 70 μm nylon filter (Becton, Dickinson and Company, Franklin Lakes, NJ, USA), and the remaining tissues were discarded. The digested cells were plated in a 150 cm² culture dish (Nalge Nunc International, Rochester, NY, USA) in complete culture medium (CCM): αMEM containing 10% fetal bovine serum (FBS; Invitrogen), 100 units/ml penicillin (Invitrogen), 100 μg/ml streptomycin (Invitrogen), and 250 ng/ml amphotericin B (Invitrogen) and incubated at 37°C with 5% humidified CO₂. The medium was changed to remove nonadherent cells one day later and cultured for 14 days as passage 0, then replated at 100 cells/cm² in a 150 cm² culture dish, cultured for 14 days and cryopreserved as passage 1. To expand the cells, a frozen vial of the cells was thawed, plated in 60 cm² culture dishes, and incubated for four days in the recovery plate. These cells were replated at 100 cells/cm² in a 150 cm² culture dish (passage 3), and cultured for an additional 14 days. These passage 3 cells were harvested and used in this study.

Isolation and culture of rabbit synovial MSCs

This study was approved by the Animal Experimentation Committee of Tokyo Medical and Dental University (No.0120296A). Wild type skeletally mature Japanese White Rabbit and GFP transgenic rabbits [11,12] were anesthetized with an intramuscular injection of 25 mg/kg ketamine hydrochloride and with an intravenous injection of 45 mg/kg sodium pentobarbital and 150 μg/kg medetomidine hydrochloride. Synovium was harvested aseptically from knee joints of the rabbits, and digested in a 3 mg/ml collagenase type V in aMEM for three hours at 37°C. The digested cells were plated at 5 × 10⁴ cells/cm² in a 150 cm² culture dish in CCM and incubated at 37°C with 5% humidified CO₂. The medium was changed to remove nonadherent cells one day later and cultured for seven days as passage 0. The cells were then trypsinized, harvested and resuspended to be used for further assays. The cells that were transplanted in animals to be sacrificed at Day 0 and Day 14 were labeled for cell tracking by the fluorescent lipophilic tracer DiI (Molecular Probes, Eugene, OR, USA). For labeling, synovial MSCs were resuspended at 1 × 10⁶ cells/ml in αMEM without FBS and a DiI was added at a final concentration of 5 μl/ml. After incubation for 20 minutes at 37°C with 5% humidified CO₂, the cells were centrifuged at 450 g for 5 minutes and washed twice with phosphate-buffered saline (PBS) and the cells were then resuspended in CCM and cultured in hanging drops. We already reported that these cells had characteristics of MSCs [3,6,7,11].

Preparation of aggregates of synovial MSCs

A total of 2.5 × 10⁵ synovial MSCs were trypsinized, harvested and resuspended in 35 μl of CCM, plated on an inverted culture dish lid. The lid was inverted and placed on a culture dish containing PBS. The cells were cultured at 37°C with 5% humidified CO₂ for three days in hanging drops.

Histology of aggregates of human synovial MSCs

Aggregates of human synovial MSCs were fixed with 2.5% glutaraldehyde in 0.1 M PBS for two hours. The aggregates were washed overnight at 4°C in the same buffer and post-fixed with 1% OsO4 buffered with 0.1 M PBS for two hours. The aggregates were dehydrated in a graded series of ethanol and embedded in Epon 812. Semi-thin (1 μm) sections for light microscopy were collected on glass slides and stained for 30 seconds with toluidine blue.

In vitro chondrogenic differentiation assay

A total of 2.5 × 10⁵ human synovial MSCs cultured as a monolayer were pelleted by trypsinization and centrifugation. The pellets or aggregate of human synovial MSCs cultured for three days in hanging drops were cultured in 400 μl chondrogenic medium consisting of high-glucose Dulbecco’s modified Eagle’s medium (Invitrogen) supplemented with 1,000 ng/ml BMP-7 (Stryker Biotech, Boston, MA, USA), 10 ng/ml transforming growth factor-β3 (R&D Systems, Minneapolis, MN, USA), 100 nM dexamethasone (Sigma-Aldrich Corp., St. Louis, MO, USA), 50 μg/ml ascorbate-2-phosphate, 40 μg/ml proline, 100 μg/ml pyruvate, and 1:100 diluted ITS+Premix (6.25 μg/ml insulin, 6.25 μg/ml transferrin, 6.25 ng/ml selenious acid, 1.25 mg/ml bovine serum albumin, and 5.35 mg/ml linoleic acid; BD Biosciences Discovery Labware, Bedford, MA, USA). The medium was changed every 3 to 4 days for 21 days.

Histology of pellets of human synovial MSCs

The pellets were embedded in paraffin, cut into 5-μm sections and stained with 1% Toluidine Blue. For immunohistochemistry, sections were treated with 0.4 mg/ml proteinase K (DAKO, Carpinteria, CA, USA) in Tris-HCl and normal horse serum after deparaffinization. Primary antibodies for type II collagen (Daiichi Fine Chemical, Toyama, Japan) and a secondary antibody of biotinylated horse anti-mouse IgG (Vector Laboratories, Burlingame, CA, USA) were employed. Immunostaining was detected with VECTASTAIN ABC reagent (Vector Laboratories) followed by 3,3′-diaminobenzidine staining.

Real-time RT PCR analysis

Total RNA was extracted from human synovial MSCs in a monolayer culture, aggregates of human synovial MSCs cultured for 1, 2 and 3 days, and the pellets cultured for 7, 14 and 21 days using QIAzol (Qiagen, Hiden, Germany) and the RNeasy mini kit (Qiagen). cDNA was synthesized with oligo-dT primer from total RNA using the Transcriptor High Fidelity cDNA Synthesis kit (Roche Diagnostics) according to the manufacturer’s protocol. Reverse transcription (RT) was performed by 30 minutes incubation at 55°C followed by 5 minutes incubation at 85°C. Real-time PCR was performed in a LightCycler 480 instrument (Roche Diagnostics). Primer sequences and TaqMan probes are listed in Table 1. After an initial denaturation step (95°C for 10 minutes), amplification was performed for 40 cycles (95°C for 15 seconds, 60°C for 60 seconds). Relative amounts of mRNA were calculated and standardized as previously described [13,14].

Table 1. Real time-RT PCR primer sequences

DNA microarray analysis

Total RNA was extracted from human synovial MSCs in a monolayer culture, aggregates of human synovial MSCs cultured for three days. Human Genome U133 Plus 2.0 Array (GeneChip; Affymetrix, Santa Clara, CA, USA) containing the oligonucleotide probe set for more than 47,000 transcripts was used. The fluorescence intensity of each probe was quantified by using the GeneChip Analysis Suite 5.0 (Affymetrix). Gene expression data were normalized in Robust MultiChip Analysis (RMA). To analyze the data, we used hierarchical clustering using TIGR MultiExperiment Viewer (MeV) [15]. The microarray data have been deposited to the public database (GEO accession# GSE 31980).

In vivo transplantation

Under anesthesia, the left knee joint was approached through a medial parapatellar incision, and the patella was dislocated laterally. Full-thickness osteochondral defects (5 mm × 5 mm wide, 1.5 mm deep) were created in the trochlear groove of the femur. A total of 5, 10, 20, 40 and 80 aggregates of autologous rabbit synovial MSCs (2.5 × 10⁵ cells/aggregate) or 25 and 100 smaller aggregates of autologous rabbit synovial MSCs (1.0 × 10⁵ cells/aggregate) suspended in PBS were transplanted to the defect. To trace the transplanted cells, DiI-labeled aggregates of autologous rabbit synovial MSCs and aggregates of allogenic synovial MSCs derived from GFP transgenic rabbit were transplanted to the defect. For the control group, the defect was left empty. All rabbits were returned to their cages after the operation and were allowed to move freely. Animals were sacrificed with an overdose of sodium pentobarbital at 1, 2, and 4 days and at 12 weeks after the operation (n = 5 at each time).

Macroscopic examination

The cartilage defects were examined macroscopically for color, integrity and smoothness. Osteoarthritic changes and synovitis of the knee were also investigated. Digital images were taken using an Olympus MVX10 (Olympus, Tokyo, Japan).

Histological examination and fluorescent microscopic examination

The dissected distal femurs were immediately fixed in a 4% paraformaldehyde (PFA) solution. The specimens were decalcified in 4% ethylenediamine tetraacetic acid solution, dehydrated with a gradient ethanol series and embedded in paraffin blocks. Sagittal sections 5 μm thick were obtained from the center of each defect and were stained with toluidine blue and Safranin O. For fluorescent microscopic examination and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining, the fixed specimens were incubated at 4°C for three hours in 5%, 10%, 15% and 20% sucrose solution, respectively. After incubation, the fixed specimens were mounted on a holder. Then 30% optimal cutting temperature (OCT) (Sakura Finetek, Tokyo, Japan) in sucrose solution was added gently into the holder. The holder was frozen in hexan chilled by dry ice and stored at -80°C. Cryosections (10 μm) were prepared with an ultracut S microtome (Reichert, Wien, Austria) and a Microm HM560 cryostat.

Histological score

Histological sections of the repaired tissue were analyzed using a grading system consisting of five categories (cell morphology, morphology, matrix staining, surface regularity, cartilage thickness and integration of donor with host), which were modified from the repaired cartilage score described by Wakitani and colleagues [16], so that overly thick, regenerated cartilage could not be overestimated [6]. The scoring was performed in a blinded manner by two observers and there was no significant interobserver difference. The ratio of the safranin-O positive area over the defect was evaluated. Zeiss AxioVison software (Carl Zeiss, Oberkochen, Germany) was used for measurement of defects and safranin-O positive areas.

In vitro viability assay

Aggregates of rabbit synovial MSCs were plated at 1 or 40 aggregates/well in 96-well plates (Nunc) in CCM, and incubated at 37°C with 5% humidified CO₂ for seven days without medium change. Aggregates were fixed in 4% PFA for TUNEL staining.

TUNEL staining

For TUNEL staining, an apoptosis in situ detection kit (Wako Pure Chemical Industries, Ltd, Osaka, Japan) was used. The frozen semi-thin sections were incubated with terminal deoxynucleotidyl transferase for 10 minutes at 37°C in a moist chamber. The sections were washed with 0.1 M PBS for 15 minutes. Peroxidase-conjugated antibody was then applied to the specimens at 37°C for 10 minutes in a moist chamber. The sections were developed with 3,3-diaminobenizidine and counterstained with methyl green.

Statistical analysis

Comparisons between two groups were analyzed using the Mann-Whitney U test. Comparisons between multi groups were analyzed using the Kruskal-Wallis test and the Steel test. A P-value of < 0.05 was considered statistically significant.

Results

Appearance of aggregates of human synovial MSCs

Human synovial MSCs were aggregated using the hanging drop technique (Figure 1A). Three days after being cultured in the drop (Figure 1B), the aggregate, consisting of 250,000 MSCs, became approximately 1 mm in diameter (Figure 1C). The aggregate was not easily broken by manipulation. Sagittal sections of the aggregates showed heart-shape as a whole (Figure 1Da). The superficial layer was composed of spindle cells parallel to the surface, whereas the deep layer was comprised of round cells both at top and bottom of the aggregate (Figure 1Db, c). Though cells positive for TUNEL staining were observed, the number was only approximately under 5% (Figure 1Dd).

Figure 1. Preparation and appearance of aggregates of human synovial MSCs. (A): Scheme of preparation of aggregates using hanging drop technique. (B): Drops hanging on the cover of 15 cm dish. (C): Macroscopic image of aggregate consisted of 250,000 MSCs, three days after cultured in hanging drop. (D): Sagittal sections of aggregates stained with toluidine blue (a, b, c) and TUNEL (d). TUNEL positive cells are indicated with arrows.

Transcriptome profile of aggregates of human synovial MSCs

To examine the sequential changes of gene expression profiles during aggregation of human synovial MSCs, microarray analyses were performed. The differences of gene profile between before and after aggregation exceeded those among donor variances (Figure 2A). The number of genes up-regulated more than five-fold was 621. The number of genes up-regulated more than 100-fold was 10, and these genes were related to hypoxia (integrin, alpha 2 (ITGA2), stanniocalcin 1 (STC1), chemokine (C-X-C motif) receptor 4 (CXCR4)), nutrient (BMP2, proprotein convertase subtilisin/kexin type 1 (PCSK1), secreted phosphoprotein 1 (SPP1), ITGA2, STC1), extracellular region (MMP1, MMP3), and cell adhesion (SPP1, ITGA2) (Table 2). The most up-regulated gene was BMP2, increased to 273 folds (Table 2). STC1 was also highly up-regulated in aggregates of synovial MSCs. The number of genes down-regulated less than one-fifth was 409, and the ontology for the genes was related to cell cycle. The microarray data are available at the public database (GEO accession# GSE 31980).

Figure 2. Transcriptome changes after aggregation of human synovial MSCs. (A): Hierarchical clustering analysis for gene expression profile of aggregates of MSCs. The color code for the signal strength in the classification scheme is shown in the box left. High expression genes are indicated by shades of red and low expression genes are indicated by shades of green. (B): Expressions of chondrogenesis-related genes (SOX5, SOX6, SOX9, BMP2) and anti-inflammatory genes (TSG-6, STC-1) in aggregates of MSCs at Days 0 to 3 by real time RT-PCR analysis. The results are shown in four individual donors respectively.

Table 2. The top 10 upregulated genes in aggregates of MSCs

To further investigate gene expressions during aggregation of human synovial MSCs, real time RT-PCR analyses were additionally used for chondrogenesis-related genes (SRY (sex determining region Y)-box (SOX)5, -6, -9, and BMP2) and anti-inflammatory genes (TNFα inducible gene 6 (TSG-6), and STC-1) in four donors. In most cases, expressions for these genes increased sequentially (Figure 2B).

In vitro chondrogenesis of aggregates of human synovial MSCs

In vitro chondrogenic ability of human synovial MSCs after hanging drop culture was compared to that of MSCs after monolayer culture (Figure 3A). Aggregates of MSCs differentiated into chondrocytes as well (Figure 3B). The wet weight of pellets derived from MSCs after hanging drop culture was heavier than that of pellets derived from MSCs after monolayer culture in all four donors at 14 or 21 days (Figure 3C). Real time RT-PCR analysis showed higher expression levels of collagen (COL)2A1, aggrecan and SOX9 for pellets derived from MSCs after hanging drop culture compared to MSCs after monolayer culture at 14 and 21 days (Figure 3D). Cartilage extracellular matrix synthesis and accumulation of type II collagen were confirmed by histological analysis stained with toluidine blue and immunohistochemical analysis (Figure 3E).

Figure 3. In vitro chondrogenic ability of human synovial MSCs after hanging drop culture (A): Scheme for the analyses. (B): Macroscopic images of pellets derived from aggregates of MSCs and those of pellets derived from MSCs in a monolayer culture. (C): Wet weight in four individual donors. Values are the means with standard deviation (SD) (P < 0.05 by the Mann-Whitney U test). (D): Expressions of chondrogenesis-related genes by RT-PCR analyses. Values are the means with SD among four donors. The fold changes of SOX9 and AGGRECAN expression levels were shown when the gene expression levels at Day 0 were normalized as 1. The fold changes of COL2A1 expression levels were shown when the gene expression levels in MSCs in monolayer at Day 7 were normalized as 1 because COL2A1 expression level at Day 0 was undetectable. (E): Histological sections of pellets stained with toluidine blue and immunohistochemical analysis for type II collagen.

In vivo analysis for cartilage regeneration by transplantation of aggregates of synovial MSCs in rabbits

To examine whether transplantation of aggregates of synovial MSCs promotes cartilage regeneration, in vivo study was performed in rabbits. To further investigate the optimal number of aggregates consisting of 250,000 MSCs, 0 to 80 aggregates were transplanted into the defect.

At 0 days, in the case of 40 and 80 transplanted aggregates, the osteochondral defects were filled with aggregates labeled with DiI macroscopically (Figure 4A).

Figure 4. Cartilage regeneration by transplantation of aggregates of synovial MSCs in rabbits. (A): Macroscopic observation of osteochondral defects one minute after transplantation of indicated number of aggregates of MSCs. The aggregate consisted of 250,000 MSCs, labeled with DiI for visualization. (B): Macroscopic and histological observation. For histologies, sagittal sections were stained with safranin-O (SO) and toluidine blue (TB). (C, D): Magnified histology of the indicated area. (E): Histological score. Values are the means with SD. (n = 5; P < 0.05 by the Kruskal-Wallis test and the Steel test). (F): Ratio of the safranin-O positive area to the defect area. Values are the means with SD. (n = 5; P < 0.05 by the Kruskal-Wallis test and the Steel test).

At four weeks, in the case of 5 and 10 transplanted aggregates, the osteochondral defect was mostly covered with a thick cartilage matrix (Figure 4B, C). In the case of 20 and 40 transplanted aggregates, the defect was partially covered with cartilage matrix. In the case of 80 transplanted aggregates, the defect was filled with only fibrous tissue, which appeared to be similar to the control (Figure 4B).

At 12 weeks, in the case of 10 transplanted aggregates, the border between cartilage and bone moved up, and thickness of the regenerated cartilage became similar to the neighboring cartilage (Figure 4B, D). In the case of 5 and 20 transplanted aggregates, the bone defect was repaired, but the cartilage defect was filled partially with cartilage matrix. In the case of 40 and 80 transplanted aggregates, the osteochondral defect was poorly repaired, similar to the control (Figure 4B). Histological score was the best and the safranin-O positive area ratio was highest in the case of 10 transplanted aggregates both at 4 and 12 weeks (Figure 4E, F).

To trace MSCs, 10 aggregates of GFP positive MSCs were transplanted into the defect. At Day 1, no GFP positive aggregates could be observed in the knee joint except the defects with a fluorescent stereomicroscope. Histologically, aggregates changed their forms but have not fused yet (Figure 5A). At four weeks, the defect was filled with cartilage matrix and the GFP positive cells were still observed both at the bottom and the center of the regenerated cartilage (Figure 5B). Regenerated cartilage consisted of both GFP positive cells and GFP negative cells.

Figure 5. Transplantation of 10 aggregates of synovial MSCs derived from a GFP rabbit. (A): Sagittal sections of osteochondral defect under fluorescence for GFP at one day. (B): Macroscopic and histological observation four weeks after transplantation. Nuclei were shown as blue in higher magnified pictures.

Influences of cell number per aggregate and of aggregate number for transplantation

Cell number per aggregate as well as aggregate number may be a factor affecting properties of the aggregates. To answer this question, 25 or 100 aggregates, in which an aggregate consisted of 100,000 MSCs, were transplanted into the osteochondral defect.

At four weeks, in the case of 25 transplanted aggregates, the defect was fully filled with cartilage matrix (Figure 6A), in which the result was different from the case of 20 or more aggregates, in which an aggregate consisted of 250,000 MSCs. In the case of 100 transplanted aggregates, the defect was filled with fibrous tissue, and the histological score was inferior and the safranin-O positive area ratio was smaller. (Figure 6B, C).

Figure 6. Influences of cell number per an aggregate and of aggregate number for transplantation. (A): Macroscopic and histological observation four weeks after transplantation of 25 or 100 aggregates in which an aggregate consisted of 100,000 MSCs. (B): Histological score. Values are the means with SD (n = 4; P < 0.05 by the Mann-Whitney U test). (C): Ratio of the safranin-O positive area to the defect area. Values are the means with SD. (n = 4; P < 0.05 by the Mann-Whitney U test). (D): Histological observation two weeks after transplantation of 10 and 80 aggregates in which an aggregate consisted of 250,000 MSCs labeled with DiI. Sagittal sections under fluorescence and the serial sections stained with TUNEL were shown. (E): In vitro analyses of aggregates of rabbit synovial MSCs. One or 40 aggregates, in which an aggregate consisted of 250,000 MSCs, were cultured in a well of 96-well plates. Macroscopic images for the wells and sagittal sections of the aggregates stained with TUNEL were shown.

Influences of aggregate number on viability of MSCs

To clarify why transplantation of aggregates over a certain number resulted in poor outcome, viability of cells was first examined by TUNEL staining. Compared to the case of 10 transplanted aggregates, much more TUNEL positive cells could be observed in the case of 80 transplanted aggregates (Figure 6D).

Another factor might be a nutrient deprivation and in vitro analyses using aggregates of rabbit synovial MSCs were performed. Seven days after 1 or 40 aggregates were cultured in a well of 96-well plates, the medium color changed to yellow in the case of 40 aggregates, while the color remained red in the case of only 1 aggregate (Figure 6E). TUNEL positive cells were much higher in the case of 40 aggregates than in the case of only 1 aggregate.

Discussion

In this study, to form aggregates of synovial MSCs, the hanging drop technique was used [8-10]. This is a simple method; expensive or specific tools are not required. Three days after cultured in the drop, the aggregate, consisting of 250,000 MSCs, became approximately 1 mm in diameter, large enough to be visible and solid enough to aspirate with a pipette. Aggregates of MSCs sank faster in the suspension medium than dispersed MSCs and helped to avoid loss of MSCs from targeted cartilage defect. The use of aggregates was practically convenient for transplantation of MSCs.

In the previous report, the number of apoptotic or necrotic cells was greater in aggregates prepared with 100,000 or 250,000 human bone marrow MSCs, which was examined by flow cytometry, measuring propidium iodide uptake and annexin V labeling [10]. We examined the viability of aggregates of MSCs by TUNEL staining and confirmed that cells positive for TUNEL staining were observed; the number was small compared to the previous report. This difference may have been due to the difference of methods. Microarray analysis showed up-regulation of genes with ontology for regulation of cell death. The microarray data are available at the public database (GEO accession# GSE 31980). These results suggest that aggregation of 250,000 MSCs affect the viability of cells. However, we thought that aggregates of MSCs could be used as a source for cartilage regeneration because most cells which are cultured in drops for three days are viable.

Aggregation of synovial MSCs changed the gene expression profile dramatically without any special tools or chemical factors. This is possibly due to environmental changes, including cell-to-cell contact, hypoxic condition and low nutrient condition. Aggregation of human synovial MSCs increased expressions of several chondrogenesis-related genes and the most up-regulated gene was BMP2, which was also up-regulated in bone marrow MSCs [8,10].

In this study, we compared in vitro chondrogenesis potential of synovial MSCs after hanging drop culture with that of MSCs after monolayer culture. We used 1,000 ng/ml BMP7 for in vitro chondrogenic differentiation assay. We previously examined the dose effect of BMP6 between 0 to 500 ng/ml for in vitro chondrogenesis of bone marrow MSCs. Cartilage pellets increased in size along with the concentration of BMP6, and a maximal effect was at 500 ng/ml [17]. Our preliminary experiments showed that 1,000 ng/ml BMP6 induced larger cartilage pellets than 500 ng/ml BMP6 in bone marrow and synovial MSCs. We obtained similar results with BMP7. Real time RT-PCR analysis showed higher expression levels of COL2A1, aggrecan and SOX9 for pellets derived from MSC-aggregates after hanging drop culture compared to those of MSCs in a monolayer culture. Furthermore, the wet weight of pellets derived from MSC-aggregates after hanging drop culture was heavier than that of pellets derived MSCs in a monolayer culture. These indicate that chondrogenic potential increased in aggregates of MSCs after hanging drop culture.

In this study, we used an osteochondral defect model of rabbits, which have a higher, self-renewal capacity than bigger animals and humans. Therefore, the results obtained here should be critically evaluated. However, we prepared negative controls, which healed poorly at 4 and 12 weeks. We previously confirmed that the osteochondral defect created in the trochlear groove of the femur, similar to this study, was not repaired without any treatments 24 weeks after surgery [6]. These findings indicate that this rabbit model is useful to evaluate the effects of the treatments for cartilage regeneration.

For in vivo analysis of cartilage regeneration by transplantation of aggregates of synovial MSCs in rabbits, successful cartilage regeneration was observed in the cases of a relatively small number of transplanted aggregates of MSCs, and the worst results were observed when the highest number of aggregates of MSCs was transplanted. These results were not what we expected, because we previously reported that better cartilage regeneration was obtained when higher cell densities of MSCs were embedded in collagen gel [3].

Why were poor results obtained when more than a certain number of aggregates were transplanted? We listed three possible reasons. First, nutrition to maintain transplanted MSCs was depleted and the environment around transplanted MSCs worsened when too many aggregates were transplanted. As shown in Figure 6E, in the case of 40 aggregates that were cultured for seven days in a well of 96-well plates, medium color changed to yellow. This means that adjustment of pH could not be controlled. Second, TUNEL positive cells increased when too many aggregates were transplanted. The number of TUNEL positive cells was higher when too many aggregates were transplanted (Figure 6D) than before transplantation (Figure 1D) and after a suitable number of aggregates were transplanted (Figure 6D). Third, transplantation of too many aggregates prevented chondro-progenitor cells from moving to the osteochondral defect from bone marrow and from synovial fluid.

We confirmed that transplanted aggregates of synovial MSCs were directly differentiated into chondrocytes by transplanting MSCs derived from GFP transgenic rabbit. This result suggests that aggregates of synovial MSCs were involved in the reparative process. However, as shown in Figure 5B, in the case of aggregates of GFP positive MSCs being transplanted, regenerated cartilage consisted of both GFP positive cells and GFP negative cells. MSCs existed in synovial fluid [18] and these MSCs contributed to the repair of cartilage injury [6,19]. These results suggest that some host MSCs were also involved in the reparative process. In addition, host MSCs may have been involved in the anti-inflammatory process. In our rabbit osteochondral defect model, inflammation like a synovitis was not severe even in the control group. Therefore, we could not confirm the anti-inflammatory effect of MSCs. It would be interesting to investigate the anti-inflammatory effect of transplantation of aggregates of synovial MSCs and host MSCs in other arthritis models.

As previously reported, in bone marrow MSCs [10], aggregates of human synovial MSCs expressed anti-inflammatory genes TSG6 and STC1. TSG6 is secreted by synoviocytes, mononuclear cells and chondrocytes under inflammatory conditions and has an anti-inflammatory effect. Overexpression of TSG6 or administration of recombinant TSG6 inhibited inflammation and joint destruction in a murine collagen induced arthritis model [20-23]. STC1 is reported to have an anti-apoptotic effect as well as an anti-inflammatory effect [24,25]. However, their roles in joint homeostasis are unknown.

In this study, transplantation of low numbers of aggregates, in other words, low density of aggregates to the volume of the cartilage defect, showed better regeneration (Figures 4 and 6). This is favorable for clinical application. We have performed clinical trials of autologous human synovial MSCs transplantation for cartilage defects. In the experiences of 12 patients, approximately 50 million synovial MSCs at passage 0 were transplanted for approximately 280 mm² cartilage defects (unpublished data). In a rabbit model, we transplanted synovial MSC-aggregates into the osteochondral defects without any loss of cells, and 10 MSC-aggregates (2.5 × 10⁶ cells) per 25 mm² defects were needed for better cartilage regeneration. According to these data, we can prepare a sufficient amount of human synovial MSCs at passage 0.

In this study, we did not use scaffolds for transplantation of aggregates of synovial MSCs. We were able to adhere aggregates of synovial MSCs on the osteochondral defect without scaffolds; however, the use of scaffolds or materials to improve survival of transplanted cells is attractive. One of the methods is the use of a fibrin glue, which has an effect of improving survival of transplanted cells [26]. In addition, cell transplantation of MSCs with a fibrin glue can probably be performed under arthroscopic surgery. Further studies are needed to improve cell transplantation procedures.

Conclusion

Aggregated synovial MSCs were a useful source for cartilage regeneration considering such factors as easy preparation, higher chondrogenic potential and efficient attachment.

Abbreviations

αMEM: α-minimal essential medium; BMP: bone morphogenetic protein; CCM: complete culture medium; COL: collagen; CXCR4: chemokine (C-X-C motif) receptor 4; EDTA: ethylenediaminetetraacetate; FBS: fetal bovine serum; GFP: green fluorescent protein; GJB2: gap junction protein, beta 2; ITGA2: integrin, alpha 2; MeV: MultiExperiment Viewer; MMP: matrix metalloproteinase; MSC: mesenchymal stem cell; OCT: optimal cutting temperature; PBS: phosphate-buffered saline; PCSK1: proprotein convertase subtilisin/kexin type 1; PFA: paraformaldehyde; RMA: Robust MultiChip Analysis; RT: reverse transcription; SD: standard deviation; SO: safranin-O; SOX: SRY (sex determining region Y)-box; SPP1: secreted phosphoprotein 1; STC1: stanniocalcin 1; TB: toluidine blue; TFPI2: tissue factor pathway inhibitor 2; TNF: tumor necrosis factor; TSG6: TNFα inducible gene 6; TUNEL: terminal deoxynucleotidyl transferase dUTP nick end labeling.

Competing interests

The authors declare that they have no competing interests.

Authors’ contributions

SS participated in the design of the study, carried out the animal experiments, analyzed the results and drafted the manuscript. TM participated in the design of the study and provided the administrative and financial support. KT participated in the design of the study. SI helped with histological analysis. HM and AU carried out the microarray analysis and participated in the evaluation of the results. IS participated in the design of the study, provided the financial support and completed the final manuscript. All authors read and approved the final manuscript.

Acknowledgements

This study was supported by “the Project for Realization of Regenerative Medicine” and “the Global Center of Excellence (GCOE) Program” by the Ministry of Education, Culture, Sports, Science and Technology (MEXT), Japan to IS, grants from the Japanese Ministry of Education Global Center of Excellence Program, International Research Center for Molecular Science in Tooth and Bone Diseases to TM, and from a Health and Labor Sciences Research Grant, Research on Regenerative Medicine for Clinical Application to IS. Recombinant human BMP-7 was distributed by Stryker Biotech. We thank Miyoko Ojima for her expert help with histology and Izumi Nakagawa for management of our laboratory.

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