The effects of pulsed low-intensity ultrasound on chondrocyte viability, proliferation, gene expression and matrix production

Me: This shows one of the studies done testing the possibility of using the LIPUS technology to increase the chondrocyte proliferation. In this study though it is called PLIUS, not LIPUS as we have called it but they are the same thing. Again I note that this study is critical in showing whether using LIPUS is even worth looking deeper into. From only being able to get to the abstract, my opinion on the technology is mixed. PLIUS was shown to inhibit the expression of type X collagen. It seems that using the lower level of PLIUS intensity of 2 mW/cm^2 seem to inhibit chondrocyte hypertrophy which is something that we desire since chondrocyte hypertrophy means that they are no longer in the zone to proliferate which is something we don’t want. We want to hold off on any type of Collagen Type X production as much as possible to increase the number of chondrocytes available to be in the hypertrophy zone to increase in size. 
Original contribution

The effects of pulsed low-intensity ultrasound on chondrocyteviability, proliferation, gene expression and matrix production

  • Zi-Jun Zhang*, James Huckle, Clair A Francomano*, Richard G.S Spencer*,
  • * National Institutes of Health, National Institute on Aging, Baltimore, MD, USA
  •  Smith and Nephew Group Research Centre, York Science Park, Heslington, York, UK
  • http://dx.doi.org/10.1016/j.ultrasmedbio.2003.08.011, How to Cite or Link Using DOI
  • Permissions & Reprints

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Abstract

This study was designed to examine the effects of pulsed low-intensity ultrasound (PLIUS) on chondrocyteviability, proliferation, matrix production and gene expression. Chondrocytes were isolated from the distal part of the sternum of 16-day-old chick embryos and cultured in alginate beads. PLIUS at 2 mW/cm2 (group PLIUS2) and 30 mW/cm2 (group PLIUS30) was applied to chondrocytes for a single 20-min treatment. A control group was treated without PLIUS. The viability of chondrocytes was not affected by exposure to PLIUS. PLIUS influenced chondrocyte proliferation in an intensity-dependent manner. By day 7 after application of PLIUS, the gene expression and synthesis of aggrecan was the same as in the controls. At this same time point, the expression and synthesis of type II collagen was not different between the controls and PLIUS30, but was increased in PLIUS2. PLIUS was shown to inhibit the expression of type X collagen. This inhibition of chondrocyte hypertrophy may prove to be significant in the management of cartilage degeneration. (E-mail: spencer@helix.nih.gov)

Dietary Lactose Improves Endochondral Growth and Bone Development And Mineralization

Me: It would seem that from just reading this neat little abstract that the common food product of milk may actually be causing kids to grow taller from not just increased Vitamin D and Calcium intake and density. It seems that the Lactose sugar themselves also help with endochondral growth. I am even willing to make the daring hypothesis that it could be possible that lactose could be the real ingredient in milk that causes kids to grow and maybe it is not from the Vitamin D and Calcium, since there have been many studies done that refuted and confirmed the age old thinking that Milk will increase a kids growing rate and height from the Calcium and Vitamin D intake. However, it is important to know that there is more in milk that can cause growth and height increase than what was previously believed to be in it.
J Nutr. 1988 Jan;118(1):72-7.

Dietary lactose improves endochondral growth and bone development and mineralization in rats fed a vitamin D-deficient diet.

Miller SC, Miller MA, Omura TH.

Source

Division of Radiobiology, School of Medicine, University of Utah, Salt Lake City 84112.

Abstract

Lactose promotes the intestinal absorption of calcium independent of the vitamin D endocrine system. The purpose of this study was to determine the effect of lactose supplementation on endochondral bone growth, bone development and mineralization in weanling rats fed a vitamin D-deficient diet. Rat pups were weaned from vitamin D-deficient dams and fed a vitamin D-deficient diet containing sucrose as the primary carbohydrate source or a similar diet but containing 20% lactose. After 4 wk, body weights, serum calcium levels and endochondral bone elongation rates in the lactose-fed animals were higher than in rats fed the sucrose diet. In addition, bone weights, bone calcium content, percent bone ash of bone dry weight, percent metaphyseal osseous tissues and bone osteoid content in the lactose-fed rats were different from those in the rats fed the sucrose diet. In all cases the changes in osseous tissues that were observed in the animals fed the lactose-supplemented diet were toward normal values as observed in age-matched animals fed a vitamin D-replete diet. The improvements in bone growth and development due to lactose supplementation occurred independent of the vitamin D endocrine system and are likely the result of improved calcium absorption in the intestine.

PMID: 3335941      [PubMed – indexed for MEDLINE] 

 

How Lateral Synovial Joint Loading Works To Increase Height From Non-Distraction: FAQs and Concerns Answered (Guest Post)

Me: I have been having this issue for the longest time with any method or technique that does not involve at least comes kind of  distraction in the bones we want to stretch and lengthening because I have been having the hardest time wrapping my head around how such methods could possibly work. I have talked to my coworker about this and Tyler but the answer was always “chondrogenesis” which I couldn’t understand. Finally I decided to send Tyler an email so he can clarify what that means. Also, I wanted to ask him about this specific issue when applied to his method, the LSJL, and why it works.

Questions

Inbox…

Natural Height Growth, naturalheightgrowth@gmail.com,  to Tyler,  Sep 30 (2 days ago)

My coworker and i are having a discussion on a main point. how is lsjl able to break apart and stretch out the hard inorganic calcium phosphate bone matrix of the cortical bone even if chondrocytes are being created at such a high level. it should not do anything since you are pushing against something that is harder than concrete.

Tyler Davis to me, Oct 1 (1 day ago)

LSJL doesn’t try to push apart the bones.  LSJL tries to induce chondrogenesis in the epiphysis of the bone.  The chondroinductive properties of LSJL have been validated by gene expression results and histology diagrams.  The ability for hydrostatic pressure, fluid flow, and LIPUS to induce chondrogenesis of adult human MSC’s has also been validated in vivo.  LSJL induces similar stimuli as HP, FF, and LIPUS

Natural Height Growth naturalheightgrowth@gmail.com, to Tyler,  2:12 AM (19 hours ago)

i still don’t get how creating more cartilage cells in the epiphysis will lead to longitudinal growth. even if this type of external stimuli is inducing the right type of genes to produce and release the right type of protein, how can they get around the hard bone? the epiphysis may not be as hard as the diaphysis but it is still very strong.

People keep telling me that the answer is in the chondrogenesis like her but I can’t figure it out. Here is my thinking…

1. So you produce a lot of chondrocytes from MSC differentiation in the cancellous external cavity in the epiphysis ends….

2. The loading (assume it is lsjl method) causes certain genes in the MSC and other HSC in the bone marrow to up regulate and release growth hormones and increase rate of changing into the right type of
3. Some BMPs, FGF, and maybe some other growth factors which are proteins and hormones increases in formation and output or gets turned on
How does the epiphysis increase in length? I realize now that my focus or at least what is easier for me to understand is the orthopedics part, less of the endocrine stuff, and least of all the genetics stuff. So I am focusing on the orthopedics right now.
You say that you are trying to induce the generation and proliferation of chondrocytes in the inner cavity of the epiphysis. How does the chondrocytes then make the overall bone longer? The epiphysis still has a very hard surface so the cell should not be able to pass through the outer bone but then it has the peristeum to deal with. If you remember, the growth plate passes completely through the long bone for it to separate the two bone parts further from each other.
The only explanation I can think of is that you are assuming that the constant rate of osteoblast and osteoclast formation and resorption on the outside and inside might get the inner produced chondrocytes to be pushed out of the inner cavity and once it reaches close to the surface, it causes a bone lengthening process similar tot eh growth plate. However, growth plates have chondrocytes stacked neatly on top of each other. Please explain the process because I am stuck over and over again. the science from a engineering and physics point of view makes no sense.
this is the same kind of problem i am having with fully understanding the ilizarov method. I am still confused whether the fracture is made completely through the bone, thus opening up to the bone marrow cavity or is it just the outer dense bone that is cracked open.

maybe i am being really stupid here so call me “billy bob” but this one part makes no sense.

Tyler David to me, 2:43 AM (19 hours ago)

The principal is that cartilage is capable of growth from within whereas bone is not.  Growth plates have to be capable of pushing incredible amounts of weight like an elephant for example.  Bone can only grow on the outside whereas cartilage is capable of interestitial growth.  Therefore the growth plate doesn’t have to be completely from one end to the bone to the other.  The periosteum is capable of lengthening as well.

If you look at this picture of a finger fracture: http://www.heightquest.com/2010/02/empirical-evidence-of-possibility-of.html.  You can see that my finger increased in length(diagonally) without the whole finger being broken.

And LSJL increases matrix degradation via MMP3, MMP2, and MMP14.  Thus addressing the cortical bone problem.

Growth plate chondrocytes are often not stacked neatly on top of each other.  Also the epiphysis is porous with trabecular bone.  It has a lot of empty spaces filled with marrow.  Yellow/Red doesn’t matter because adiposal stem cells are capable of chondrogenic differentiation too.

Cartilagenous growth plates are capable of interstitial growth.  Growth plates always work against force as it is trapped between two bone ends.  Thus, growth plates should be able to generate growth from within a bone.  One issue is that static compression decreases growth plates and as you say bones are heavy.  So after inducing chondrogenesis with say LSJL it may be best to use microgravity afterwards say inversion or just giving your legs a break for an hour.  Inversion is hard to do for long periods of time though.

In summary: Growth plates can beat forces by definition as they are always trapped between a rock and a hard place(epiphysis and diaphysis).  The sides of the bone are just another force.

Feel free to include this conversation as a “guest post” just include the backlink.

————————-

Natural Height Growth, naturalheightgrowth@gmail.com, to Tyler, 1:30 PM (8 hours ago)

Okay. Now I am finally starting to reach some kind of understanding on what you mean. However, there is still two small points that I haven’t figured out yet.

1. So you increase the chondrocytes. The chondrocytes release the collagen type ii and proteoglycan which is supposed to form a cartilage matrix.

Am i supposed to assume that cartilage is being formed in the epiphysis cavity from doing these loadings?

If you say yes, then the problem is that I have never heard of an incidence where cartilage is actually being formed inside the marrow cavity.

I have always thought that it requires chondrocytes and cartilage matrix to push apart bone, not just chondrocytes. don’t you need cartilage matrix to push the bones.?

in principle and theory, your idea does make sense.

From a physics point of view, if you can get the chondrocytes to multiply, and multiply some more , they will push. The forces produced in the epiphysis will defintiely increase the hydrostatic pressure.

However the force/area which is pressure on the surface area of the saivty will be divided and distributed across the entire thing.

We both know that long bones have high compressive force and tensile strength so force exerted on the axis directions will have to be large. You are pressing laterally from the inside and they say the pressure needed at least from the outside inwards direction is only 50 MPa.

I am visualizing a picture of the femur in my mind as a cylinder with two spheres on the end, like a dumbbell shape. the cortical bone will be strong so the forces there won’t do much. Nature wants to take the path of least resistance so the hydrostatic pressure and chondrocytes will try to put a stretch on the area which is the weakest, which should than be in the epiphysis, but in the lateral direction.

With my logic, this means that LSJL should make the epiphysis ends wide, but only a little bit longer in on the axis which we want, which is longitudinally. Your theory makes sense but the affects will be so small like an exact few millimeters in long bone lengthening.

2. The other main concern which I see is that the original growth plates were not encapsulated like the new chondrocytes. I agree that the original growth plates did have a lot of pressure exerted on them. growth plates are only a few millimeters and they have to support and hold up a 200 lb person so there is a lot of force/ sqr inch. they are pushing up and down. but the sides are not clamped shut.

I will say this again, the chondrocytes produced are pushing in a closed system which is surrounding by upwards of 1 cm thick of bone that is concrete in strength. and the way the pressure will force upon the inner ways will be distributed in a way that goes towards the weaker epiphysis sides.

I stated before that the tensile strength of long bone is 150 MPa. this means the chondrocytes needed to push up to at least 70% of this amount to cause some deformation. They also have to push in the right direction, along the axis. they are trapped in 1 cm thick physically mature adult femur bones.

If I can figure out or calculate how much force/area the original growth plates had to deal with, and it comes out to be anything close to 50-100 MPa then LSJL will definitely work to increase long bones longitudinally. if the values are not close, then I would guess most of the bone changes would be towards increased epiphysis thickness, and very little for real lengthening.  You will get some bone lengthening but little.

I just need to do more research to see what the biomechanical values are of the bones and growth plates. can you explain away my two main concerns?

Tyler Davis to me, 2:01 AM (11 hours ago)

Unfortunately there haven’t been a lot of studies on forces required to lengthen bone or I haven’t come across them.  There’s a study that I mention here http://www.heightquest.com/2011/05/why-does-hydrostatic-pressure-induce.html about mitotic cell rounding and

The idea is that differentiating chondrocytes will automatically secrete matrix.

Unfortunately, I can’t explain away your concerns now.  Ideally we’d need to the growth plates in action live to see what forces they generate.

I have done research myself on the biomechanical forces of the bone and growth plates and couldn’t find very much.

So unfortunately I can’t explain away your concerns at this time.

Natural Height Growth, naturalheightgrowth@gmail.com, to Tyler, Oct 3 (2 days ago)

Okay. I’ll take this conversation and turn it into another post with the back link. It won’t be posted until I get through the protein pathway and endocrinology posts which could take up to 2 weeks. Im just getting around to doing all the reading and research.

The Height Increase And Grow Taller Clinics Kiness, Hamsoa, And Seojung Growth Clinic In South Korea

Me: It seems that the South Koreans have had these height increasing and growth taller clinic appearing everywhere now, especially in the capital of Seoul. The competitive nature for jobs and future spouses causes many parents to put a lot of financial investment to make their kids taller. There is 3 clinics mentioned in the article, Kiness, Hamsoa, and Seojung Growth Clinic. One of the people who run the clinic says that there are now 36 clinics also opened in China in collaboration to make other people taller. It is just amazing to see what the East Asian people are willing to go through to get taller.

I am going to try to get in contact with any of the 3 clinics and see if I can strike up a partnership with them and get them to promote this website or I can promote these clinics if they can show what goes on inside their clinics and see what types of methods and techniques they are doing to increase the kids height. If they would like me to promote their clinics, I have to make sure these techniques really work and have shown through careful scrutiny and analysis that everything is safe.


The article below was found from the New York Times article HERE

South Korea Stretches Standards for Success

Woohae Cho for The International Herald Tribune

Trainer Choi Hyong-jin helped Kang Hyon-sung, 5, and his sister, Kang Hyon-hee, 7, as they tried a special treadmill during a growth-spurring exercise at the Seojung growth clinic in Seoul.

By CHOE SANG-HUN
Published: December 22, 2009

SEOUL, South Korea — With acupuncture needles trembling from the corners of her mouth like cat’s whiskers, Moon Bo-in, 5, whined with fear. But the doctor, wearing a yellow gown patterned with cartoon characters, poked more needles into her wrists and scalp.

Kim Ok-hee and her daughter, Kang Hyon-hee, 7, at the Seojung growth clinic in Seoul.

“It’s O.K., dear,” said her mother, Seo Hye-kyong. “It will help make you pretty and tall. It will make you Cinderella.”

Swayed by the increasingly popular conviction that height is crucial to success, South Korean parents are trying all manner of remedies to increase their children’s stature, spawning hundreds of growth clinics that offer hormone shots, traditional Eastern treatments and special exercises.

“In our society, it’s all about looks,” said Ms. Seo, 35. “I’m afraid my daughter is shorter than her peers. I don’t want her to be ridiculed and lose self-confidence because of her height.”

Ms. Seo spends $770 a month on treatments for her daughter and her 4-year-old son at one such clinic, Hamsoa, which has 50 branches across the country and offers a mix of acupuncture, aromatherapy and a twice-a-day tonic that contains deer antler, ginseng and other medicinal herbs.

“Parents would rather add 10 centimeters to their children’s stature than bequeath them one billion won,” said Dr. Shin Dong-gil, a Hamsoa doctor, invoking a figure in Korean currency equal to about $850,000. “If you think of a child as a tree, what we try to do here is to provide it with the right soil, the right wind, the right sunshine to help it grow. We help kids regain their appetite, sleep well and stay fit so they can grow better.”

Koreans used to value what was perceived as a grittiness on the part of shorter people. “A smaller pepper is hotter,” according to a saying here, and one need look no further for proof than to the former South Korean strongman Park Chung-hee, or across the demilitarized zone to the North Korean ruler Kim Jong-il, who claims to be 5-foot-5 (but adds inches with elevator shoes and a bouffant hairstyle).

But smaller is no longer considered better, thanks in part to the proliferation of Western models of beauty and success. “Nowadays, children scoff if you mention Napoleon and Park Chung-hee,” said Park Ki-won, who runs the Seojung Growth Clinic. “On TV, all young pop idols are tall. Given our society’s strong tendency to fit into the group and follow the trend, being short is a problem. Short kids are ostracized.”

Concerns about the trend are growing, too, with some groups warning that growth clinics, while operating within the limits of the law, promise far more than the evidence supports.

Yoon Myoung, a top researcher at Consumers Korea, a civic group that, with the help of scientists, has been investigating the clinics, said parents should be more skeptical.

“There is no clinical proof or other evidence that these treatments really work,” Ms. Yoon said. “They use exaggerated and deceptive ads to lure parents. But Korean families often have only one child and want to do whatever they can for that child.”

Last month, the simmering discomfort over the trend exploded when a college student put it into blunt words on national television.

“Being tall means being competitive,” Lee Do-kyong, a student at Hongik University in Seoul, said on a television talk show. “I think short guys are losers.”

Bloggers vilified her, and lawmakers denounced the station, KBS-TV, for not editing her comments. Viewers filed defamation lawsuits. Ms. Lee was forced to apologize, and the Communications Standards Commission ordered the show’s producers to be reprimanded for “violating human rights” and “stoking the looks-are-everything phenomenon.”

“She simply said what everyone thinks but doesn’t dare say in public,” said Dr. Kim Yang-soo, who runs a growth clinic called Kiness. “Here, if you change your height, you can change your fate.”

At his clinic, Kim Se-hyun, a fifth grader, walked on a treadmill with her torso encased in a harness suspended from an overhead steel bar. The contraption, the clinic maintains, will stretch her spine and let her exercise with less pressure on her legs.

Nearby, sweat rolled off Lee Dong-hyun, 13, as he pedaled a recumbent bicycle while reading a comic book. Behind him, his sister, Chae-won, the shortest girl in her first-grade class, stretched to touch her toes on a blue yoga mat, squealing as an instructor pushed down against her back.

Two years ago, their mother, Yoon Ji-young, had tried giving Dong-hyun growth hormone shots, which have also increased in popularity here. But many doctors will prescribe them only for exceptionally small children with severe growth disorders. And parents have been discouraged by their high cost and fears of side effects.

Ms. Yoon said she was spending $850 a month on the shots but stopped after eight months.

Now she drives her children to Kiness three times a week. “Both my husband and I are short,” said Ms. Yoon, 31, who is about 5 feet tall. “I don’t want my children to blame us for being short when they grow up.”

Another mother at the clinic, Chang Young-hee, 54 and 4-foot-10, said her children had already experienced height discrimination. Both her daughters are college graduates and have good jobs, but when they reached marrying age, matchmakers regarded their short stature as a defect.

“It felt like a blow to the head,” Ms. Chang said. “I learned a lesson. If you fall behind in your studies, you can catch up later. But if you miss the time to grow, you miss it forever.”

Her daughters eventually married, but for the past four years, she has been taking her youngest child, Seo Dong-joon, to Kiness. The boy, now 15, knows his goal.

“If I’m tall, I’ll have an advantage selecting my future wife,” he said, holding an English vocabulary book, which he studies while exercising. “Short guys are teased at school.”

South Koreans have been growing taller anyway, thanks to changes in their diet. Over the past 30 years the average height of high school senior boys in South Korea has increased 3.5 inches, to 5-foot-8, according to government data. Senior girls grew an average of 2 inches, to 5-foot-3.

Doctors at the growth clinics say that most children simply aspire to the new average height, but with more tall teenagers, those who are not as tall seem even shorter. “The gap between tall and short has become more pronounced,” said Dr. Park of Seojung, who recently opened 36 joint-venture growth clinics in China and said the quest to become taller was regionwide.

If so, one country that has been left behind is North Korea. Food shortages there have left children stunted, according to the United Nations and private relief agencies. Dr. Park cited the case of a 16-year-old who fled North Korea last July to join his mother, who had arrived in the South three years earlier. The boy was 5 feet tall, almost four inches below the South Korean average.

“His height wasn’t unusual for the North,” Dr. Park said. “But when his mother saw him again, she cried because the boy hadn’t grown at all, and because she knew the disadvantages he’d face here.”

“My dream is to open growth clinics in North Korea,” Dr. Park said, “so that, once we unify, children from both sides will be able to stand shoulder to shoulder, not with one side a head taller than the other.”

Characterization of the Distinct Orthotopic Bone-Forming Activity of 14 BMPs

Me: Here is my interpretation on this really important article. If you want to get bones and cartilage to grow fast, use either a BMP2 & BMP6 mixture or a BMP2 & BMP9 mixture. The BMP6 and BMP9 has shown to have the most bone formation ability while the BMP2 is needed for the BMP6 to work. Plus, it seems that only the BMP2 or BMP7 has the ability to generate chondrocytes, which the BMP6 and the BMP9 seem to lack. The key seems to be to use a combination BMP formula if we ever plan to do growth factor injections with a compound mixture. 

Gene Therapy (2004) 11, 1312–1320. doi:10.1038/sj.gt.3302298; Published online 22 July 2004, (source HERE)

Characterization of the distinct orthotopic bone-forming activity of 14 BMPs using recombinant adenovirus-mediated gene delivery

Q Kang1,2,5, M H Sun1,5, H Cheng1,5, Y Peng1,3, A G Montag1,4, A T Deyrup1,4,6, W Jiang1, H H Luu1, J Luo1, J P Szatkowski1, P Vanichakarn1, J Y Park1, Y Li1, R C Haydon1 and T-C He1,3

  1. 1Molecular Oncology Laboratory, Department of Surgery, The University of Chicago Medical Center, Chicago, IL, USA
  2. 2The Children’s Hospital of Chongqing University of Medical Sciences, Chongqing, China
  3. 3Committee on Genetics, The University of Chicago, Chicago, IL, USA
  4. 4Department of Pathology, The University of Chicago, Chicago, IL, USA

Correspondence: Dr T-C He, Molecular Oncology Laboratory, Department of Surgery, The University of Chicago Medical Center, 5841 South Maryland Avenue, MC3079, Chicago, IL 60637, USA

5QK, MHS, and HC Contributed equally to this work

6Current address: Department of Pathology, Emory University, Atlanta, GA, USA.

Received 18 August 2003; Accepted 7 February 2004; Published online 22 July 2004.

Abstract

Efficacious bone regeneration could revolutionize the clinical management of bone and musculoskeletal disorders. Although several bone morphogenetic proteins (BMPs) (mostly BMP-2 and BMP-7) have been shown to induce bone formation, it is unclear whether the currently used BMPs represent the most osteogenic ones. Until recently, comprehensive analysis of osteogenic activity of all BMPs has been hampered by the fact that recombinant proteins are either not biologically active or not available for all BMPs. In this study, we used recombinant adenoviruses expressing the 14 types of BMPs (AdBMPs), and demonstrated that, in addition to currently used BMP-2 and BMP-7, BMP-6 and BMP-9 effectively induced orthotopic ossification when either AdBMP-transduced osteoblast progenitors or the viral vectors were injected into the quadriceps of athymic mice. Radiographic and histological evaluation demonstrated that BMP-6 and BMP-9 induced the most robust and mature ossification at multiple time points. BMP-3, a negative regulator of bone formation, was shown to effectively inhibit orthotopic ossification induced by BMP-2, BMP-6, and BMP-7. However, BMP-3 exerted no inhibitory effect on BMP-9-induced bone formation, suggesting that BMP-9 may transduce osteogenic signaling differently. Our findings suggest that BMP-6 and BMP-9 may represent more effective osteogenic factors for bone regeneration.

Bone regeneration is critical to the effective management of many bone and musculoskeletal disorders, such as fracture healing, spinal fusion, and osteoporosis, which are responsible for a large portion of healthcare expenditure in the developed countries—approximately $14 billion is spent annually on treating osteoporotic fractures in the US alone.1 Bone is a highly mineralized tissue and is one of the few organs that retains the potential for regeneration in adult life. Bone also undergoes continuous remodeling throughout life.2, 3 Three major types of cells are present in bone tissues: bone-forming osteoblasts, bone-resorbing osteoclasts, and chondrocytes. Osteoblasts are derived from the mesenchymal stem cells, which also serve as precursor cells for myocytes, adipocytes, and chondrocytes. It has been known for almost half a century that demineralized bone can induce de novo bone formation.4 The molecular identity of the bone-forming factors in demineralized bone was subsequently revealed to be bone morphogenetic proteins (BMPs).5 BMPs belong to the TGFbeta superfamily, and play an important role in embryonic development and bone formation.6, 7 At least 15 types of BMPs have been identified in humans. BMP signal transduction begins via interaction with the heterodimeric complex of two transmembrane serine/threonine kinase receptors, BMPR type I and BMPR type II.8, 9 The activated receptor kinases phosphorylate the transcription factors Smads 1, 5, and/or 8. The phosphorylated Smads then form a heterodimeric complex with Smad 4 in the nucleus and activate the expression of target genes in concert with other coactivators.10, 11, 12

Although the molecular mechanisms underlying osteoblast differentiation remain to be defined, BMPs play an important role in regulating osteoblast differentiation and subsequent bone formation. Traditionally, various bone grafts have been used to promote osteogenesis in bone and musculoskeletal disorders. The identification of BMPs has generated great interest due to their potential use in bone regeneration.3 Several recombinant forms of BMPs, mostly rhBMP-2 and rhBMP-7 (a.k.a., OP-1), have been shown to induce bone formation in vivo13,14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 and both rhBMP-2 and rhBMP-7 have been tested in clinical trials.25, 26, 27 In addition to the direct application of recombinant BMP proteins, numerous reports have confirmed the ability of adenoviral or retroviral vector-mediated gene transfer of several BMPs to induce bone formation in animal models.18, 20, 21, 23, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37,38, 39, 40, 41, 42, 43, 44

Although a plethora of studies have demonstrated the ability of several BMPs, mostly BMP-2 and BMP-7, to promote osteogenesis, it is unclear whether or not BMPs other than those currently being tested in clinical trials are more potent stimulators of new bone formation. Thus, it is important to conduct a comprehensive comparative analysis of the in vivo osteogenic activity of all BMPs. This line of investigation has been hampered by the fact that recombinant proteins are either not biologically active or not available for all BMPs. We have recently constructed a panel of recombinant adenoviral vectors that express the 14 types of human BMPs (BMP-2–BMP-15).45 Recombinant adenoviral vectors are ideal for this line of investigation for following reasons.46, 47, 48, 49, 50, 51 First, adenoviral vectors can transduce osteoblast progenitor cells with high efficiency. Second, the biologically active BMPs are continuously produced inside mammalian cells. Third, BMP-mediated bone osteoblast differentiation does not require long-term expression. Fourth, adenoviral vectors can be used in both in vitro and in vivo studies. In this study, we sought to carry out a comprehensive analysis of the distinct in vivo bone-forming activity of the 14 types of human BMPs. Using an orthotopic ossification animal model, we demonstrate that BMP-2, BMP-6, and BMP-9 (BMP-7 to a lesser extent) are the most potent osteoinductive BMPs. Our findings strongly suggest that, in addition to BMP-2 and BMP-7 that are currently used in clinical trails, BMP-6 and BMP-9 could represent equally, if not more effective osteogenic factors for bone regeneration in a clinical setting.

Results

Distinct ability to induce an osteogenic marker, alkaline phosphatase (ALP), by 14 BMPs in the osteoblast progenitor C2C12 cells in vitro

In order to comprehensively analyze the distinct osteogenic activity of BMPs, we have recently constructed a panel of recombinant adenoviral vectors that express the 14 types of human BMPs, designated as AdBMPs.45 As shown in Figure 1a, the level of transgene expression of the AdBMPs were in general comparable (ie, <2-fold difference among different BMPs), as demonstrated by RT-PCR analysis. These PCR products should be specifically derived from the adenoviral vector-mediated expression (rather than from the endogenous genes), as the 3′-end primer was derived from the SV40 poly-A cassette. Using these adenoviral vectors, we have recently demonstrated that BMPs displayed a distinct ability to induce osteoblast differentiation of mesenchymal progenitor cells in vitro.45 In this study, we sought to determine the relative in vivo osteogenic activity of the 14 types of BMPs. We first tested the ability of individual AdBMPs to induce the earlier osteogenic marker alkaline phosphatase in the C2C12 line, which is myoblastic and can be trans-differentiated into osteoblast progenitors upon BMP stimulation. As shown in Figure 1b, ALP activity was remarkably induced by five of the 14 BMPs at four days after AdBMP infections. Among the five osteogenic BMPs, BMP-2, BMP-6, and BMP-9 induced the ALP activity to a much greater extent (approx. 169-, 215-, and 273-fold over the GFP control, respectively), while BMP-4 and BMP-7 increased ALP activity by 44- and 73-fold, respectively. These findings are consistent with our previous studies.45 It should be pointed out that several BMPs (eg, BMP-10, and BMP-13) also induced a 2–3-fold increase of ALP activity over the basal level under the same assay conditions.

Induction of alkaline phosphatase activity by the 14 types of human BMPs in C2C12 cells. (a) Relative level of AdBMP-mediated transgene expression. C2C12 cells were infected with AdBMPs or AdGFP for 40 h. Total RNA was isolated and converted into cDNA products by reverse transcription, which were used for RT-PCR reactions using BMP-specific primers (5′-end) and a 3′-end primer derived from SV40 polyA. Resultant products ranged from 500 to 600 bps (indicated by arrows). ‘+’, PCR products from +RT reactions of the original cDNA synthesis; ‘-‘, PCR products from -RT reactions of the original cDNA synthesis; ‘M’, 1-Kb Plus ladder from Invitrogen; ‘G’, AdGFP; ‘2–15’, AdBMP-2 to AdBMP-15. (b) Subconfluent C2C12 cells were infected with AdBMPs and the control AdGFP. At 4 days after infection, cells were lysed for colorimetric assays of alkaline phosphatase activity using p-nitrophenyl phosphate as a substrate. Representative results from at least three independent experiments are shown. See Materials and methods for details.

Orthotopic bone formation effectively induced by several but not all BMPs in athymic mice

We next sought to test the in vivo osteogenic effect of the 14 BMPs. In order to effectively assess the osteogenic ability of the BMPs, we used an orthotopic ossification animal model, in which C2C12 cells were first transduced with AdBMPs and subsequently implanted into the quadriceps muscles of athymic nude mice by intramuscular injection. Orthotopic ossification was assessed by X-ray radiography and histological evaluation at 3 and 5 weeks after injections. As illustrated in Figure 2, ossification was readily detected on X-ray radiographies from the animals injected with AdBMP-2, 6, 7, and 9-transduced C2C12 cells at 3 weeks (Figure 2a) and 5 weeks (Figure 2b). For BMP-6 and BMP-9, histologic examination at both time points (Figure 3a and b) revealed robust and highly mineralized woven bone with scattered osteoblast-rimming and occasional osteoclasts. Osteoid was also present. In addition, BMP-6 showed bone marrow elements with a range of hematopoietic cells. At the 3-week time point, BMP-2 was characterized by well-calcified foci without bone formation; however, at the 5-week time point, these foci had developed into mature bone. BMP-7, on the other hand, was shown to induce much weaker ossification. Interestingly, we failed to detect any signs of ossification in the animals injected with AdBMP-4-transduced C2C12 cells, which is surprising because we have demonstrated that BMP-4 is capable of inducing ALP activity in vitro (Figure 1b).45 These results were reproducible in two additional rounds of animal studies using different batches of AdBMP-4 preparations, which were consistently shown to induce ALP activity in C2C12 cells in vitro (data not shown). Further, our RT-PCR analysis demonstrated that the expression level of BMP-4 was comparable with that of other BMPs, especially BMP-2, BMP-6, and BMP-9 under the same assay condition (Figure 1a). Currently, we are still searching for any satisfactory explanations for this discrepancy between BMP-4’s in vitro versus in vivo osteogenic activity. Nevertheless, all of the above findings were reproducible in three batches of independent experiments. The remaining samples had no evidence of ossification at 5 weeks. The injection site in these sections demonstrated exuberant granulation tissue and reparative changes.

Histology of BMP-induced bone formation

We next examined the histology of the recovered injection sites. Overall, the histology correlated well with the findings from X-ray radiography. At the 3-week time point, BMP-2, BMP-6, BMP-7, and BMP-9 demonstrated varying degrees of ossification. BMP-2 and BMP-7 were the least developed with small foci of woven bone (Figure 3a). Both BMP-6 and BMP-9, however, had multiple foci of immature woven trabecular bone. In addition, BMP-9 demonstrated focal cartilaginous differentiation. The bone in BMP-6 and BMP-9-treated animals formed a shell-like rim around a proliferating mass of spindle-shaped C2C12 cells. The 5-week samples demonstrated increased maturation with more mature osteoid matrix and trabecular bone-like structures with accentuation of the shell-like rim, especially in BMP-6 and BMP-9-treated animals. There was less extensive ossification in the BMP-2 sections. Bone marrow elements were present in the BMP-6 sections and chondrocytes and cartilaginous matrix were increased in the BMP-9 sections (Figure 3b). Interestingly, the injected C2C12 cells formed a desmoid-like cell mass in nonosteogenic BMP injections and the GFP control. Even in the animals injected with BMP-2, BMP-6, BMP-7, and BMP-9-transduced C2C12 cells, such cell mass was still visible, and multiple ossification centers were observed at the periphery of the cell mass. BMP-2-, BMP-6-, BMP-7-, and BMP-9-induced osteogenesis was further confirmed by Masson’s Trichrome staining (Figure 3c).

Antagonistic effect of BMP-3 on BMP-induced bone formation

We sought to investigate how the osteogenic BMPs were affected by BMP-3, a known negative regulator of bone formation, as BMP-3 knockout animals exhibited an increase in bone density.52 When C2C12 cells transduced by AdBMP-3 and one of the four osteogenic AdBMPs were coinjected intramuscularly for 3 weeks, BMP-2 and BMP-6-induced ossification was completely blocked by BMP-3, and most of the BMP-7-induced ossification was inhibited by BMP-3 (Figure 4a). However, BMP-3 coinjection did not exert any effect on BMP-9-induced calcification (Figure 4a), strongly suggesting that BMP-9 may exert its osteogenic activity via a distinct signaling mechanism. Similar results were obtained for the 5-week groups (data not shown). The histological findings were consistent with those from X-ray radiographic results (Figure 4b). The three samples without ossification demonstrated C2C12 cell proliferation with entrapped skeletal muscle while the BMP-3+BMP-9 sections had multiple foci of woven trabecular bone similar to BMP-9 injection alone.

BMP-3-mediated inhibition of bone formation induced by BMP-2, BMP-6, and BMP-7, but not by BMP-9. (a) Osteogenic AdBMPs (ie, BMP-2, -6, -7, and -9)-transduced C2C12 cells were either injected alone (top row) or coinjected with AdBMP-3 (bottom row) into the right quadriceps of athymic mice. Animals were killed at 3 weeks and subjected to X-ray radiography. Ossification sites were indicated by arrows. Each experimental group contained four mice, and representative radiographies from three batches of experiments are shown. (b) Histological evaluation of the BMP-3 coinjection sites. B, osteoid matrix; C, injected C2C12 cells; and M, muscle cells. Magnification, times 150.

Bone formation induced by direct intramuscular injection of AdBMPs

Recent studies suggest that skeletal muscles may harbor pluripotent mesenchymal stem cells, including osteoblast progenitors.34, 44, 53 We next tested the osteoinductive activity of the 14 BMPs via direct intramuscular injection of AdBMPs. At the 3 and 5-week time points, we did not observe apparent ossification on X-ray radiography (data not shown). However, when the 5-week injection sites were examined histologically, various degrees of cartilaginous and/or osteoid matrix formation were observed in BMP-2, BMP-6, BMP-7, and BMP-9-injected animals (Figure 5). Samples derived from BMP-2 and, to a lesser extent, BMP-7 injection sites contained more cartilage-like chondrocyte-containing structure, while osteoid matrix and mature lamellar bone were present with evidence of bone marrow colonization and remodeling in BMP-6 and BMP-9-injected animals. Unlike in the experiments with C2C12 injections, direct intramuscular injections with AdBMPs (ie, the above-mentioned four osteogenic BMPs) induced more diffuse ossification. This may also explain why the calcification (ie, by BMP-9) was not readily detected by X-ray radiography. These findings also suggest that orthotopic osteogenesis induced by direct intramuscular injection with osteogenic AdBMPs may be less efficient than that induced by introduction of AdBMP-transduced osteoblast progenitor cells, implying that osteoblast progenitor cell-based gene therapy may be a more efficacious approach to bone regeneration, although it is possible that the reduced bone formation was resulted from the potentially less-efficient gene transfer associated with direct intramuscular injections than that with AdBMP-transduced C2C12 cells.

Discussion

Orthotopic bone formation induced by direct intramuscular injection of AdBMPs. Approximately 109 PFU of AdBMPs or AdGFP were directly injected into the quadriceps of athymic mice. Animals were killed at 5 weeks after injection and subjected to X-ray radiography (not shown). Each experimental group had four mice. Representative results from two independent experiments are shown. BMP-6-treated sample was decalcified. B, osteoid matrix; BM, bone marrow cells; Ch, chondrocytes; and M, muscle cells. Magnification, times 200.

Successful bone regeneration mediated by biofactors could revolutionize the clinical management of musculoskeletal disorders, including fracture healing and spinal fusion.54 Several biological factors, such as TGFbeta, BMP, FGF, PDGF, IGF, and LMP-1, have been investigated for their potential use in bone regeneration and skeletal repair.55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69 BMPs have been shown to be the most promising, and clinical trials with recombinant BMP-2 and -7 are ongoing.54, 70, 71, 72, 73 These BMPs have shown varying degrees of success in the clinical setting and further study on their mechanisms of action and optimal formulations is required to optimize effectiveness of this strategy for promoting osteogenesis.

To the best of our knowledge, this reported study represents the first of its kind to evaluate the in vivo osteogenic activity of BMPs in a comprehensive fashion. The observation that BMP-2 exhibited osteogenic activity in our study is consistent with early data from human clinical trials.25, 27 While BMP-7 exhibited apparent osteogenic activity, its ability to induce ossification was significantly less robust than that of BMP-2, BMP-6, and BMP-9. These findings mirror the moderate success of the rhBMP-7 (ie, OP-1) in a recent clinical trial.26

It is intriguing, however, that BMP-6 and BMP-9 emerged as the most potent inducers of orthotopic ossification in vivo. Although considerable genetic and developmental studies have been conducted to elucidate the biological functions of BMP-6, its osteogenic activity has not been investigated to any significant degree in animal studies or clinical trials. BMP-6-deficient mice are largely unremarkable, with the exception of a defect in the sternum.74 Its expression during embryogenesis is closely coupled with BMP-2, and the lack of noticeable defects in BMP-6-deficient mice may be due to functional compensation by BMP-2.74 Nevertheless, our findings corroborate well with a recent study in which BMP-6 was shown to induce the most rapid tissue calcification when compared with BMP-2 or BMP-4 in an athymic nude rat model,75 although for reasons to be determined AdBMP-4 reproducibly failed to induce orthotopic bone formation in this study.

BMP-9 is one of the least studied members of the BMP family. Originally identified from fetal mouse liver cDNA libraries, BMP-9 (a.k.a., GDF-2) is highly expressed in the developing mouse liver, and recombinant human BMP-9 (rhBMP-9) stimulates hepatocyte proliferation.76 BMP-9 has also been shown to be a potent synergistic factor for hematopoietic progenitor cell generation and colony formation,77 and may also play a role in the induction and maintenance of the neuronal cholinergic phenotype in the central nervous system.78 In addition, it has recently been shown that BMP-9 exhibits an apparent osteoinductive effect in rat models.21, 23, 79 However, the mechanisms underlying BMP-9-mediated osteogenic signaling remain to be defined. It is a very intriguing finding that BMP-9-mediated bone formation was not inhibited by BMP-3 in our studies. This result strongly suggests that BMP-9 may transduce a distinct osteogenic signaling pathway that is significantly different from that of BMP-2, BMP-6, and BMP-7. Through an expression profiling analysis, we have recently identified a group of downstream targets that may play an important role in the osteogenic BMP signaling pathway mediated by BMP-2, BMP-6, and BMP-9.80 Interestingly, while BMP-2, BMP-6, and BMP-9 induced a very similar overall gene expression pattern, the clustering analysis revealed that BMP-2 and BMP-9 exhibited a more similarly related expression pattern.80

In conclusion,

we have demonstrated the relative osteogenic ability of 14 BMPs and identified BMP-6 and BMP-9 (in addition to the currently used BMP-2 and BMP-7) as the most potent BMPs to induce orthotopic bone formation in vivo. Our results also suggest that the stem/progenitor cell-based ex vivo gene therapy may represent a more effective approach to bone regeneration. Future studies will focus on elucidating the major signaling differences among BMPs so that maximal synergy in bone formation can be achieved by combining BMPs that act through overlapping or converging signaling pathways. This line of investigation would help to elucidate the molecular mechanisms underlying bone formation and lead to the development of more efficacious approaches towards bone regeneration.

Recombinant adenoviral vectors expressing BMPs

The cDNA clones for human BMP-2, -3 (a.k.a., osteogenin), -4, -5, -6, -8 (a.k.a., OP-2), -9 (a.k.a., GDF-2), -10, -12 (a.k.a., GDF-7 or CDMP-3), and -13 (a.k.a., GDF6 or CDMP2) were kindly provided by the Genetics Institute (Cambridge, MA, USA). The coding sequences for BMP-7 (a.k.a., OP-1), -11 (a.k.a., GDF-11), -14 (a.k.a., GDF-5 or CDMP-1), and -15 (a.k.a., GDF-9) were PCR amplified from a human osteosarcoma cDNA library. The coding regions of the above BMPs were subcloned into pAdTrack-CMV, resulting in pAdTrack-BMPs; recombinant adenoviruses expressing BMPs (ie, AdBMPs) were subsequently generated as previously described. Continue reading

Increase Height Through Surgical Method By Cartilage Harvesting And Chondrocyte Implantation With Growth Factor Injections

So this is my first attempt at developing a real surgical strategy and process to use all of the knowledge i have learned up to this point to increase height.

Some things to note:

It turns out from rabbit studies that removal of the proliferative and hypertrophic layer in a growth plate but leaving the rest zone still there causes the proliferative and hypertrophic layer to regrow back. If you remove the rest zone layer however, you can’t regrow that back. This suggests that as long as you can create a rest zone type of chondrogenic structure, you can essentially regrow a growth plate over again.

It seems that the original growth plates were formed from the collapse and pressing together of the primary ossification center of the diaphysis and the secondary ossification center of the epiphysis. This give me (and maybe others ) the idea that to recreate a real growth plate over, you need to have two sets of growth occurring at the some time, close enough to press the two perichondrium layers (of the two growing ossification centers) together. It could be that the key to creating synthetic growth plate formation as close to the original thing is to

From Wikipedia on the perichondrium…

The perichondrium is a layer of dense irregular connective tissue which surrounds the cartilage of developing bone. It consists of two separate layers: an outer fibrous layer and inner chondrogenic layer. The fibrous layer contains fibroblasts, which produce collagenous fibers. The chondrogenic layer remains undifferentiated and can form chondroblasts or chondrocytes. Perichondrium can be found around the perimeter of elastic cartilage and hyaline cartilage. Fibrocartilage and articular cartilage both lack perichondrium. Perichondrium is a type of Irregular Collagenous Ordinary Connective Tissue, and also functions in the growth and repair of cartilage. Once vascularized, the perichondrium becomes the periosteum.

The key maybe is to press two layers of perichondrium together while still keeping the amount of pressure needed to be exerted by the sub-perichondrium bone growth in the radial direction boy the osteoblast’s activity.

Note: I am extremely unsure at this point about the difference between Type I collagen and Type II collagen and which goes where in the life cycle of the chondrocyte and the hyaline cartilage because I seem to be getting contradictory information from different sources. 

So the real strategy.

This is derived from studying the procedure of how the surgical process of Autologous Chondrocyte Implantation and Autologous Matrix-Induced Chondrogenesis

1. You first collect cartilage samples from an area of the body that has hyaline cartilage, but of the same type as the articular cartilage at the end of long bones The best places I can think of is the nose, ears, other areas like the intercondylar notch or the superior ridge of the medial or lateral femoral condyle of the patient.

2. You remove the matrix of the cartilage using enzymes and then isolate the chondrocytes.

3. Grow the chondrocytes in vitro in a specialized lab. This will take 4-6 weeks. Create enough chondrocytes to do a re-implant.

4. Create an initial distraction in the bone. This will be similar to the hammer and chisel tool and application used on the ilizarov method. Only the cortical layer is fractured, by about 1-2 millimeters in thickness.

4.5 Drill holes into the cortical bone so that the mesenchymal and progenitor cells can reach the chondrocyte implant.

These 4 elements are needed to create cartilage (from my studies).

  • progenitor cells
  • mesenchymal stem cells
  • cytokines
  • growth factors

Let’s assume that the top 5 growth factors we need at BMP2, BMP7, some FGF (not sure which type), IHH, and IGF-1. Can we make a mixture or stable compound well enough to inject into the implant later?

The waste or excretion of chondrocytes are two compounds, type II collagen and proteoglycan. Hyaline cartilage matrix is mostly made up of type II collagen and Chondroitin sulfate, both of which are also found in elastic cartilage.

5. You also add a bilayer collagen as a sort of scaffold or structure for the cartilage to growth along and form into. This will be a Collagen Type I and Collagen Type II layer. one over the other. One of the layers will be hard and dense, while the other is less hard.

6. Inject the growth factors.

7. Put a brace over the fracture to hold it in place. Similar to the ilizarove method. Wait until the bones fuse with the newly grown cartilage.

8. Once they are fused, you have to do physical therapy to make sure that the newly formed cartilage layer can deal with the weight loading when the entire human weight is applied to it. Over time the growth factors are continuously added to the cartilage region.

This is going to another one of those posts that I realize I am going to continuously edit upon to revise and make better. I really do believe that we already have the technology to increase height using a faster, less invasive, and better way to increase height through non-limb lengthening methods.

Update 1: We should also add the use of LIPUS into the method. Also, it is found that BMP6 and BMP9 may be slightly better than BMP2 and BMP7 on certain parts.