A Study On Hypogonadotropic Hypogonadism And Hypergonadotropic Hypogonadism

Me: When I was doing research on the effects of combining Gonadotropin releasing hormone analogues (GnRH-A) with human growth hormones (hGH) as a way to treat the many diverse forms of short stature, this very interesting disorder really caught my attention and I wanted to devote some time to learn more about this disorder since it might help us remove some confusion over certain issues. We know that the control and flow of the sex hormones is what can ultimately determine out final height since we have already seen how certain people without the right types of hormones or hormone receptors never even reach puberty or don’t have closed growth plates resulting in continued growth even into their 30s.

Analysis & Interpretation:

There is two main conditions we are finding with very similar names, except that one process is the opposite of the other. One is called Hypogonadotropic Hypogonadism and the other is called Hypergonadotropic Hypogonadism. The prefix “hyper” has always meant “excessive” in common english terms while the prefix “hypo” means “not enough” in common english terms. From the two Wikipedia articles I have posted below, the summarize idea is that HH and HH, the hyper and the hypo, both results in hypogonadism, which is where the body doesn’t get enough hormones from the gonads, the reproductive organs. The hypogonadotropic hypogonadism sees an impaired secretion of the gonadotropins like the FSH and LH by the pituitary gland and the hypothalamus of the brain. Since the endocrine system works from a top–>down approach, this will eventually lead to less sex hormones produced in the gonads of the person. The is why the 2nd term is hypogonadism.

The hypergonadotropic hypogonadism in comparison has the pituitary gland and the hypothalamus being just fine in function and releasing the neccessary amount of gonadotropins like the FSH and the LH but the problem is that the gonads can’t seem to be able to receive the gonadotropins or can’t process them correctly to release the high enough levels of gonad hormones, like the androgen and estrogen. The symptoms of hypogonadism in general is low sex drive, infertility, and not puberty signs like hair growth, etc. To treat the first type, where the brain areas don’t release enough, physicians can directly add the needed hormones using a GnRH agonist or a gonadotropin formulation. To treat the 2nd type, the physician can just add synthetic androgens as a hormone therapy to get the level of sex hormones in the body correct.

The connection with height increase with the two conditions is that from many genetic disorders and cases, we find that people who have no sensitivity to the sex hormones like the androgens or estrogen have often led to delayed puberty, and thus resulted in a later age for growth plate cartilage closure. There are at least 3 cases of males who had no receptors for the estrogen in their growth plates and they resulted in being very tall with the cartilage still existing in late adulthood.

From my personal analysis, it would seem that the case for tall stature can only be found with people who suffer from hypergonadotropic hypogonadism, NOT hypogonadotropic hypogonadism. I would guess that the lack of ability to release the FSH and the LP by the hypothalamic-pituitary connection would also cause insufficient release of the growth hormones too. So it would make logical sense then that the human body which can release growth hormones but stop the process of reeleasing the testosterone and estrogens which will lead to both puberty/increased growth rate but also growth plate closure means that the person who suffers only from hypergonadotropic hypogonadism will note that they never went through the great growth spurt that their peers did in adolescent, noticed that they were more likely on the short side while young, but as they grew older, they did not stop growing completely like their peers but eventually surpasses in height of their peers in their late 20s due to their open growth plate cartilage.

From the Wikipedia article on it HERE…

Hypogonadotropic hypogonadism (HH), also known as secondary or central hypogonadism, as well as gonadotropin-releasing hormone deficiency or gonadotropin deficiency (GD), is a condition which is characterized by hypogonadism due to an impaired secretion of gonadotropins, including follicle-stimulating hormone (FSH) and luteinizing hormone (LH), by the pituitary gland in the brain, and in turn decreased gonadotropin levels and a resultant lack of sex steroid production.

Causes

The type of HH, based on its cause, may be classified as either primary or secondary. Primary HH, also called isolated HH, is responsible for only a small subset of cases of HH, and is characterized by an otherwise normal function and anatomy of the hypothalamus and anterior pituitary. It is caused by congenital syndromes such as Kallmann syndrome and gonadotropin-releasing hormone (GnRH) insensitivity. Secondary HH, also known as acquired or syndromic HH, is far more common than primary HH, and is responsible for most cases of the condition. It has a multitude of different causes, including brain orpituitary tumors, pituitary apoplexy, head trauma, ingestion of certain drugs, and certain systemic diseases and syndromes.

Symptoms

Examples of symptoms of hypogonadism include delayed, reduced, or absent puberty, low libido, and infertility.

Treatment

Treatment of HH may consist of administration of either a GnRH agonist or a gonadotropin formulation in the case of primary HH and treatment of the root cause (e.g., a tumor) of the symptoms in the case of secondary HH. Alternatively, hormone replacement therapy with androgens and estrogens in males and females, respectively, may be employed.

Now let’s look at the opposite of it which is termed Hypergonadotropic Hypogonadism

From the Wikipedia article on it HERE…

Hypergonadotropic hypogonadism (HH), also known as primary or peripheral/gonadal hypogonadism, is a condition which is characterized by hypogonadism due to an impaired response of the gonads to the gonadotropins, follicle-stimulating hormone (FSH) and luteinizing hormone (LH), and in turn a lack of sex steroid production and elevated gonadotropin levels (as an attempt of compensation by the body). HH may present as either congenital or acquired, but the majority of cases are of the former nature.

Causes

Chromosomal abnormalities (resulting in gonadal dysgenesis) – Turner’s syndrome, Klinefelter’s syndrome, Swyer’s syndrome, XX gonadal dysgenesis, and mosaicism.
Defects in the enzymes involved in the gonadal biosynthesis of the sex hormones – 17α-hydroxylase deficiency, 17,20-lyase deficiency, 17β-hydroxysteroid dehydrogenase III deficiency, and lipoid congenital adrenal hyperplasia.
Gonadotropin resistance (e.g., due to inactivating mutations in the gonadotropin receptors) – Leydig cell hypoplasia (or insensitivity to LH) in males, FSH insensitivity in females, and LH and FSH resistance due to mutations in the GNAS gene (termed pseudohypoparathyroidism type 1A).

Acquired causes (due to damage to or dysfunction of the gonads) include gonadal torsion, vanishing/anorchia, orchitis, premature ovarian failure, ovarian resistance syndrome, trauma, surgery, autoimmunity,chemotherapy, radiation, infections (e.g., sexually-transmitted diseases), toxins (e.g., endocrine disruptors), and drugs (e.g., antiandrogens, opioids, alcohol).

Symptoms

Examples of symptoms of hypogonadism include delayed, reduced, or absent puberty, low libido, and infertility.

Treatment

Treatment of HH is usually with hormone replacement therapy, consisting of androgen and estrogen administration in males and females, respectively.

Axial Tibial Compression Stimulated A Robust Endocortical And Periosteal Bone-Formation Response, Maybe LSJL Works?

Me: This study is one of those studies which make me believe that the LSJL method may actually indeed work in causing some form of long bone lengthening. Sure, there are some obvious clear differences between the two ideas. The biggest one is that while this study involved axial compression on the long bone, the LSJL loading method involved loading and compression in a lateral direction. Right off the bat the researchers admit that with again, the skeleton lose its ability to respond to mechanical stimuli, so at least they get that quick rebuttal in theory arguement taken care of. They are hypothesizing that the loading would be less responsive but we already know that (so why are they doing this experiment??). From both the aged and young mice, if we load on the mid diaphysis area of the long bones, there is an analbolic response from the periosteal side (outside layer) and the endocortical side (inside layer). They say that with increase peak force, the bone formation was higher. Apparently aged mice had a far bigger response to the loading than young mice at least for the endocortical (inside) layer which surprised the researchers. The results…”Responses at the periosteal surface did not differ between age groups (p > .05). The loading-induced increase in bone formation resulted in increased cortical area in both age groups (loaded versus control, p < .05).” They conclude with..”In summary, 1 week of daily tibial compression stimulated a robust endocortical and periosteal bone-formation response at the mid-diaphysis in both young-adult and aged male BALB/c mice. We conclude that aging does not limit the short-term anabolic response of cortical bone to mechanical stimulation in our animal model.

Implications: This does make me wonder whether LSJL might really do work since we can use a thought experiment (like how einstein used to do). Let’s imagine that the long bone is surrounding by some thicker layer of cortical bone even in the epiphysis. If the compression at a axial direction on the middle diaphysis area results in the same rate of periosteal growth in both aged and young mice, then the compression of the epiphysis may actually in crease bone lengthening because the periosteum wraps around the entire long bone and even underneath the articular cartilage of the. The loading of the epiphysis would lead the bones to react by causing the peristeal layer to grow appositionally on the epiphysis which means that the axial ends do indeed get thicker and thicker which means the bones really do get longer so height is increased. Age seems to have no effect in decreased bone sensitivity, at least for the initial beginning loading. It might be that what you do from the beginning of LSJL program is what can result in the most bone/ periosteal growth lengthening, or at least until you might stop doing it for a while and go back to it a few weeks later allowing for the bone sensitivity to come back, if it ever truly do.

From PubMed study link HERE…

J Bone Miner Res. 2010 Sep;25(9):2006-15.

Aged mice have enhanced endocortical response and normal periosteal response compared with young-adult mice following 1 week of axial tibial compression.

Brodt MD, Silva MJ.

Source

Department of Orthopaedic Surgery, Washington University, School of Medicine, St Louis, MO 63110, USA.

Abstract

With aging, the skeleton may lose its ability to respond to positive mechanical stimuli. We hypothesized that aged mice are less responsive to loading than young-adult mice. We subjected aged (22 months) and young-adult (7 months) BALB/c male mice to daily bouts of axial tibial compression for 1 week and evaluated cortical and trabecular responses using micro-computed tomography (µCT) and dynamic histomorphometry. The right legs of 95 mice were loaded for 60 rest-inserted cycles per day to 8, 10, or 12 N peak force (generating mid-diaphyseal strains of 900 to 1900 µε endocortically and 1400 to 3100 µε periosteally). At the mid-diaphysis, mice from both age groups showed a strong anabolic response on the endocortex (Ec) and periosteum (Ps) [Ec.MS/BS and Ps.MS/BS: loaded (right) versus control (left), p < .05]. Generally, bone formation increased with increasing peak force. At the endocortical surface, contrary to our hypothesis, aged mice had a significantly greater response to loading than young-adult mice (Ec.MS/BS and Ec.BFR/BS: 22 months versus 7 months, p < .001). Responses at the periosteal surface did not differ between age groups (p > .05). The loading-induced increase in bone formation resulted in increased cortical area in both age groups (loaded versus control, p < .05). In contrast to the strong cortical response, loading only weakly stimulated trabecular bone formation. Serial (in vivo) µCT examinations at the proximal metaphysis revealed that loading caused a loss of trabecular bone in 7-month-old mice, whereas it appeared to prevent bone loss in 22-month-old mice. In summary, 1 week of daily tibial compression stimulated a robust endocortical and periosteal bone-formation response at the mid-diaphysis in both young-adult and aged male BALB/c mice. We conclude that aging does not limit the short-term anabolic response of cortical bone to mechanical stimulation in our animal model.

PMID: 20499381 [PubMed – indexed for MEDLINE]
PMCID: PMC3153404

Increase Height And Grow Taller Using Indian Aryuveda Homeopathic Medicine Capsules (Interesting)

This is something which I found today while I was going around the internet checking links. It is from a site HerbalCureIndia.Com. What is really interesting is that this webpage actually provides a height increase compound mixture which is really surprising. What I wanted to do is maybe go through this list and see what are the English, or scientific, or chemical names for these compounds. This is a list of minerals and plants you combine their extracts together to form a height increasing aryuveda homeopathic medicine capsule.

Ingredients

Withinia somniferra 75 mg
Puraria tuberose 75 mg
Lepidium sativum 150 mg
Gentiana sativum 150 mg
Acacia Arabica 50 mg
Ephedra gerardiana 5 mg
Cassia tora 30 mg
Oroxylum indicum 30 mg
Mucana pruries 50 mg
Cassytha filiformis 10 mg
Lauh bhasam 10 mg

One thing that has already caught my eye is the mucana pruries which I have looked into before (velvet beans) which does have a link to extra HGH release and L-Dopa. It should be spelled actually Mucuna pruriens. From the wikipedia article on the pruriens HERE…

Pharmacology

M.pruriens seeds contain high concentrations of levodopa, a direct precursor of the neurotransmitter dopamine. It has long been used in traditional Ayurvedic Indian medicine for diseases includingParkinson’s disease. In large amounts (e.g. 30 g dose), it has been shown to be as effective as pure levodopa/carbidopa in the treatment of Parkinson’s disease, but no data on long-term efficacy and tolerability are available.

In addition to levodopa, it contains serotonin (5-HT), 5-HTP, nicotine, N,N-DMT (DMT), bufotenine, and 5-MeO-DMT. As such, it could potentially have psychedelic effects, and it has purportedly been used in ayahuasca preparations.

The mature seeds of the plant contain about 3.1-6.1% L-DOPA, with trace amounts of 5-hydroxytryptamine (serotonin), nicotine, DMT-n-oxide, bufotenine, 5-MeO-DMT-n-oxide, and beta-carboline. One study using 36 samples of the seeds found no tryptamines present in them.

The leaves contain about 0.5% L-DOPA, 0.006% dimethyltryptamine (DMT), 0.0025% 5-MeO-DMT and 0.003% DMT n-oxide.The ethanolic extract of leaves of Mucuna pruriens possesses anticataleptic and antiepileptic effect in albino rats. Dopamine and serotonin may have a role in such activity.

Me: This is the actual ingredients of the capsule which is very interesting! If I even do a quick search on the first plant/mineral on the list Withinia somniferra, it turns up a PubMed study (link HERE). Another name for it is ashwagandha. From another study (source HERE) it seems that ashwagandha can reverse Alzheimer’s Disease Pathology suggesting it does it by enhancing low-density lipoprotein receptor-related protein in liver

Altern Med Rev. 2000 Aug;5(4):334-46.

Scientific basis for the therapeutic use of Withania somnifera (ashwagandha): a review.

Mishra LC, Singh BB, Dagenais S.

Source

Los Angeles College of Chiropractic (LACC), 16200 E Amber Valley Dr., Whittier, CA 90609-1166. lakshmimishra@lacc.edu

Abstract

OBJECTIVE:

The objective of this paper is to review the literature regarding Withania somnifera (ashwagandha, WS) a commonly used herb in Ayurvedic medicine. Specifically, the literature was reviewed for articles pertaining to chemical properties, therapeutic benefits, and toxicity.

DESIGN:

This review is in a narrative format and consists of all publications relevant to ashwagandha that were identified by the authors through a systematic search of major computerized medical databases; no statistical pooling of results or evaluation of the quality of the studies was performed due to the widely different methods employed by each study.

RESULTS:

Studies indicate ashwagandha possesses anti-inflammatory, antitumor, antistress, antioxidant, immunomodulatory, hemopoietic, and rejuvenating properties. It also appears to exert a positive influence on the endocrine, cardiopulmonary, and central nervous systems. The mechanisms of action for these properties are not fully understood. Toxicity studies reveal that ashwagandha appears to be a safe compound.

CONCLUSION:

Preliminary studies have found various constituents of ashwagandha exhibit a variety of therapeutic effects with little or no associated toxicity. These results are very encouraging and indicate this herb should be studied more extensively to confirm these results and reveal other potential therapeutic effects. Clinical trials using ashwagandha for a variety of conditions should also be conducted.

PMID: 10956379 [PubMed – indexed for MEDLINE] Free full text

Me: What is below is from the webpage which is linked above.

Increase your Height Growth Naturally

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Ingredients

Withinia somniferra 75 mg
Puraria tuberose 75 mg
Lepidium sativum 150 mg
Gentiana sativum 150 mg
Acacia Arabica 50 mg
Ephedra gerardiana 5 mg
Cassia tora 30 mg
Oroxylum indicum 30 mg
Mucana pruries 50 mg
Cassytha filiformis 10 mg
Lauh bhasam 10 mg

Height Increasing Capsules is one of the solution that helps in stimulating the human growth hormone that will help you in gaining the height. Human growth hormone is secreted from the anterior aspect of the pituitary gland (the master gland) situated in the brain. Production of growth hormone is at the highest in the teens (13 to 19 years of age) and this is the time maximum people attain their height and growth. But growth hormone secretions drastically falls after this age and the growth stops.

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As per the human growth medical research of Dr Ichiro Kawaguchi of Tokyo Research Laboratory of the National Health Department and Dr A. Kawata from the Kyoto University, height is not indomitable by genetic structure alone but is strongly inclined by the effects of specific hormones on the enlargement of the 26 skeletal bones and the cartilages of the 62 bones of the lower portion of the body. The excitement of the pituitary gland that will increase secretion of growth hormonal that will initiate the growth of the cartilaginous portions of the bones of the lower body, eventually leading to height increase.

Rigorous scientific research has proven that most young adults can still grow a few inches taller even after the bones in their lower body have turn into bony tissue (commonly known as “bone plate is fused”). This is because in addition to the length of the femur bone (thighbone), shinbone, and other bones in the lower body, the length of the spinal column in the upper body also significantly addto human height (about 35% of the total height).

Anatomically human spinal cord consists of 33 bone segments known as vertebrae detained together by ligaments (tough and fibrous tissue). Out of these 33 vertebrae, only the lowest 9 vertebrae are merged into two immovable bones, the sacrum and the coccyx, forming the back of the pelvis. All the other 24 vertebrae are enduringly movable and thus will never get fused (until some deformity). These 24 vertebrae are the 7 cervical (neck), 12 thoracic (back of chest), and 5 lumbar (loin).

Situated between each of these twenty four vertebrae bones are cartilage pads known as disks . The thickness of the disks establishes the length of the spinal cord and unswervingly influencing the height. There are totally twenty five such disks, their mutual length accounts twenty five percent of the total height. As these disks never fuse, they continuously grow thic Increase your height ker under the spur of growth hormone all our life.

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Recent Questions

Is there any herbal Supplement To Increase Height?

I am a 15 years old female. I have come across lots and lots of pills that claim to increase height. But most of them are scams. So hopefully in this website, I can find something that really works. Thanks!. Dovey, Toronto, Canada

Answer – Hello Dovey, Before going to any medication, one should understand the basics behind low height of an individual. The first and foremost reason of low height is nutritional deficiency and mal nutrition in the body. If a person is not consuming nutritive food of optimum grade in the right levels, he or she may fall prey to poor growth. This eventually affects the growth hormone activity in the body.

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Injecting Fibroblast Growth Factor FGF Into Fracture Sites From Distraction

Me: These types of studies are really the best type for us to see how they would effect the bones. From the 1st study, we see that from just adding 1 microgram of aFGF either every other day or every day we can get larger callus size, greater collagen content, more DNAm and also soft cartilagous callus. The calluses for the aFGF injected are bigger for at least 4 weeks until it was taken over by trabecular bone. However there was evidence from the histological testing that the mRNA expression for certain types of procollagen was lower.

From study three, we have just one injection of 100 microgram of basic FGF (bFGF) which increases the Collagen Type X and Type II mRNA expression (in hypertrophic and proliferative chondroctyes respectively) and increase the proliferation of chondroprogenitor cells in fracture callus, and thus contributes to the formation of a larger cartilage. This does not cause the ossification and maturation of the chondrocytes though. The healing process has not been decreased. In terms of height increase, this is a fascinating growth factor because it does not cause the ossification to overcome the new chondrocytes too quickly. Since maturation and ossification is the irriversible proess hypertrophic chondrocytes turn into the eventual bone, what we should be doing is maximizing the number of chondrocytes but also delaying the time of maturation.

From study two, we have the experiment repeated with one injection of 100 microgram of bFGF encapsulated in 200 microliter of fibrin gel. The results and evaluated parameters of the bone union rate, bone mineral density (BMD), and mechanical properties (strength and stiffness) of the callus was no different between the control group and the FGF injected group. the mRNA expression was also not changed much between the two groups. Again, the author states that the FGF makes the callus from the distraction larger but the healing process is not accelerated.

From PubMed study link HERE

J Orthop Res. 1990 May;8(3):364-71.

Acidic fibroblast growth factor (aFGF) injection stimulates cartilage enlargement and inhibits cartilage gene expression in rat fracture healing.

Jingushi S, Heydemann A, Kana SK, Macey LR, Bolander ME.

Source

Orthopaedic Research Unit, NIAMS, NIH, Bethesda, MD 20892.

Abstract

The effect of the administration of acidic fibroblast growth factor (aFGF) on normal fracture healing was examined in a rat fracture model. One microgram of aFGF was injected into the fracture site between the first and the ninth day after fracture either every other day or every day. aFGF-injected calluses were significantly larger than control calluses, although this does not imply an increased mechanical strength of the callus. Histology showed a marked increase in the size of the cartilaginous soft callus. Total DNA and collagen content in the cartilaginous portion of the aFGF-injected calluses were greater than those of controls, although the collagen content/DNA content ratio was not different between the aFGF-injected and control calluses. Fracture calluses injected with aFGF remained larger than controls until 4 weeks after fracture. The enlarged cartilaginous portion of the aFGF-injected calluses seen at 10 days after fracture was replaced by trabecular bone at 3 and 4 weeks. Northern blot analysis of total cellular RNA extracted separately from the cartilaginous soft callus and the bony hard callus showed decreased expression of type II procollagen and proteoglycan core protein mRNA in the aFGF-injected calluses when compared with controls. A slight decrease in types I and III procollagen mRNA expression was also observed. We concluded that aFGF injections induced cartilage enlargement and decreased mRNA expression for type II procollagen and proteoglycan core protein.

PMID: 2324855 [PubMed – indexed for MEDLINE]

From PubMed study link HERE

Calcif Tissue Int. 2007 Aug;81(2):132-8. Epub 2007 Jul 19.

Effects of a single percutaneous injection of basic fibroblast growth factor on the healing of a closed femoral shaft fracture in the rat.

Nakajima F, Nakajima A, Ogasawara A, Moriya H, Yamazaki M.

Source

Department of Orthopedic Surgery, Chiba University Graduate School of Medicine, 1-8-1 Inohana, Chuo-ku, Chiba, 260-8677, Japan.

Abstract

Recently, bioactive agents to stimulate bone formation have been available in the orthopedic field. We have shown previously that a single, local injection of basic fibroblast growth factor (bFGF) contributes to the formation of a larger cartilage (soft callus) but does not promote replacement of the cartilage by osseous tissue during experimental closed femoral fracture healing. Aiming at a clinical application, the present study was undertaken to clarify the effects of locally injected bFGF on bone (hard callus) formation and the mechanical properties of the callus in closed fracture healing in rats. Immediately after fracture, a carrier (200 muL of fibrin gel) containing 100 mug of bFGF or carrier alone was applied to the fracture site. At days 42 and 56 postfracture, the bone union rate, bone mineral density (BMD), and mechanical properties (strength and stiffness) of the callus were evaluated. Unexpectedly, with the exception of reduced stiffness in the FGF-injected callus at day 56, none of these parameters showed a significant difference between the control and the FGF-injected groups. Furthermore, the temporal expression pattern of OPN mRNA during healing was very similar between groups. We conclude that, in the healing of closed fractures of long bones, administration of bFGF forms a larger callus but does not necessarily accelerate the healing process.

PMID: 17638037 [PubMed – indexed for MEDLINE]

From PubMed study link HERE

J Orthop Res. 2001 Sep;19(5):935-44.

Spatial and temporal gene expression in chondrogenesis during fracture healing and the effects of basic fibroblast growth factor.

Nakajima F, Ogasawara A, Goto K, Moriya H, Ninomiya Y, Einhorn TA, Yamazaki M.

Source

Department of Orthopaedic Surgery, Chiba University School of Medicine, Japan.

Abstract

Chondrogenesis is an essential component of endochondral fracture healing, though the molecular and cellular events by which it is regulated have not been fully elucidated. In this study, we used a rat model of closed fracture healing to determine the spatial and temporal expression of genes for cartilage-specific collagens. Furthermore, to determine the effects of basic fibroblast growth factor (bFGF) on chondrogenesis in fracture healing, we injected 100 microg recombinant human bFGF into the fracture site immediately after fracture. In normal calluses, pro-alpha1(II) collagen mRNA (COL2A1) was detected in proliferative chondrocytes beginning on day 4 after the fracture, and pro-alpha1(X) collagen mRNA (COL10A1) in hypertrophic chondrocytes beginning on day 7. In FGF-injected calluses, the cartilage enlarged in size significantly. On day 14, both COL2A1- and COL10A1-expressing cells were more widely distributed, and the amounts of COL2A1 and COL10A1 mRNAs were both approximately 2-fold increased when compared with uninjected fractures. Temporal patterns of expression for these genes were, however, identical to those found in normal calluses. The number of proliferating cell nuclear antigen-positive cells was increased in the non-cartilaginous area in the bFGF-injected calluses by day 4. The present molecular analyses demonstrate that a single injection of bFGF enhances the proliferation of chondroprogenitor cells in fracture callus, and thus contributes to the formation of a larger cartilage. However, maturation of chondrocytes and replacement of the cartilage by osseous tissue are not enhanced by exogenous bFGF, and this results in the prolonged cartilaginous callus phase. We conclude that, in the healing of closed fractures of long bones, exogenous bFGF has a capacity to enlarge the cartilaginous calluses, but not to induce more rapid healing.

PMID: 11562144 [PubMed – indexed for MEDLINE]

Bioengineered Cartilage Pellets And LIPUS For Longtitudinal Growth (Huge Breakthrough!)

Me: This is one of the only studies I’ve found so far which seems to be talking about the exact same thing we at this website have bene talking for the longest time. I know that this article has been looked at by Tyler on HeightQuest.Com HERE and I have looked at his analysis and critique very thoroughly since this study is as close to a real study on height increase as I have found so far. It has also been looked at on other height increase forums. I can’t find the complete article but Tyler seemed to have more than me.

This article is fascinating since it would appear that the rabbits actually had their leg bones fractured, then had both a bioengineering cartilage pellet, and the LIPUS technology administered.

Huge Breakthrough 1: The study states conclusively that if we decided to use a height increase method which involved distraction, using the LIPUS technology doesn’t help. Very useful tip here. Tyler argues over the mechanics about whether they might have administered it wrong but let’s assume these Ph. Ds knew what they were doing for the time being.

Huge Breakthrough 2: The study showed that a bioengineering chondrocyte pellet was implanted in an induced distraction/fracture area. This means that somehow these group of researchers from Hong Kong have finally actually managed to completely grow a growth plate cartilage in vitro which works. The reasoning I wanted to use is this. I thought from the previous post that these researchers could only develop hyaline cartilage but this study showed that they hav also been successful in making growth plate cartilage. if you tried implanting just ordinary hyaline cartilage in growth plate physis fracture you would not get the same type of longitudinal growth because their is no perichondrium with the blood vessels going through it. You need to have a functional growth plate to allow for that type of increase in longitudinal growth, which some thing like an articular cartilage extract would not be able to do.

Implications: This shows the first real non limb-lengthening procedure for bone lengthening ever. I know that some people might want to disagree since this is just an implant into the physeal (growth plate) injury but I am extremely confident that what the researchers have done is create a synthetic, immunologically non-resistant growth plate which can be implanted back into bone and grow.

[I would be willing to guess that the growing process they use to do the growth pellet formation was from this study HERE, which Tyler also cited in the same article post from february of 2012 which I linked above. And I have the complete study article on my post. ]

From PubMed study link HERE

J Biomed Mater Res B Appl Biomater. 2011 Oct;99(1):36-44. doi: 10.1002/jbm.b.31869. Epub 2011 Jun 16.

Restoration of longitudinal growth by bioengineered cartilage pellet in physeal injury is not affected by low intensity pulsed ultrasound.

Chow SK, Lee KM, Qin L, Leung KS, Cheung WH.

Source

Department of Orthopaedics and Traumatology, Prince of Wales Hospital, The Chinese University of Hong Kong, Shatin, Hong Kong.

Abstract

Physeal fracture is a common pediatric fracture that would result in premature physeal closure in long bones, and there is currently no gold standard for its management. In this study, we investigated the application of a Bioengineered Cartilage Pellet (BCP) in repairing a rabbit physeal fracture model, and the possible effects of Low Intensity Pulsed Ultrasound (LIPUS) treatment. Rabbits with physeal fracture created were assigned to the NC group (no BCP, no LIPUS), GC group (BCP, no LIPUS), and GT group (BCP and LIPUS). Femoral lengths and cartilage area were assessed at 4, 8, and 16 weeks post-defect. After transplantation, the BCP showed continuous growth in the host and demonstrated resemblance to a natural growth plate. The GC group showed 34.1, 32.1, and 41.1% advantage in lengthening over the NC group and the GT group showed 51.1, 41.6, and 26.9% improved lengthening than the NC group, at 4 (p = 0.203), 8 (p = 0.543) and 16 weeks (p = 0.049), respectively. Cartilage area was shown to be significantly higher in GC and GT group compared to NC group (p < 0.05). No significant difference was found between GC and GT group. Femoral longitudinal growth was shown to be improved by the BCP, however no additional enhancement effect was shown to be provided by LIPUS.

PMID: 21681954 [PubMed – indexed for MEDLINE]

Hyaline Cartilage Engineered By Chondrocytes In Pellet Culture Compared To Cartilage Implants (Important)

Me: This is one of those studies which will be critical if we ever choose to do a more invasive method to increase height possibly through using implants from in vitro cultured chondrocyte pellets. I have always been slightly worried over the idea of whether the cartilage the researchers can grow and develop in the cell dish can be as similar to the native cartilage and this study has given me definite proof that from using condensation of chondrocytes in cultured pellet, with the right mediums can create the real thing. This is one huge step towards showing that regrowing an entire new growth plate is possible. However it is important to remember that although a hyaline cartilage was regrown to almost perfect in vivo statues, it is not growth plate hyaline cartilage. There is still a big difference.

Here is what I always suggest the reader do: Read the 1. abstract, 2. introduction, 3. discussion, and 4. conclusion!

Analysis: It seems that if you extract some cartilage and try to regrow the chondrocyte in the extraction, if it is a monolayer it turns into a type of fibroblastic morphology. It starts releasing Collagen type I, instead of type II and it’s aggrecan and collagen type II core protein disappears. The process of chondrocyte condensation can actually reverse the differentiation process when in some type of cell medium. This seems to be due to the cell density being decreased in the monolayer. If you put the chondrocytes in a packed 3-D form of pellet, the chondrocyte cell density is kept high which keeps it from reversing into the differentiation process and condensing.

From the study the researchers stated that for the grown culture to be the same as the native culture, they have to be structurally and biochemically the same. The grown material was tested immunohistochemically using the different types of collegan and aggrecan. From the discusssions section “After 1–2 weeks in culture, the neocartilage shared similarities with native cartilage with regard to chondrocyte phenotype, matrix distribution and the ultrastructure of collagen fibrils.”

The last sentence of the study abstract is what made me realize that we are one more step closer to a real height increase solution…”In conclusion, hyaline cartilage engineered by chondrocytes in pellet culture, without the transformation of cell phenotypes and scaffold materials, shares similarities with native cartilage in cellular distribution, matrix composition and density, and ultrastructure.”

Note: Tyler, you said in February 2012 where you analyzed this same study that you didn’t have access to the entire study but you can find it now here on this website.

From PubMed study link HERE…

J Anat. 2004 September; 205(3): 229–237.

doi: 10.1111/j.0021-8782.2004.00327.x

PMCID: PMC1571343

Hyaline cartilage engineered by chondrocytes in pellet culture: histological, immunohistochemical and ultrastructural analysis in comparison with cartilage explants

Zijun Zhang,¹ J Michael McCaffery,² Richard G S Spencer,¹ and Clair A Francomano¹

Abstract

Cartilage engineering is a strategic experimental goal for the treatment of multiple joint diseases. Based on the process of embryonic chondrogenesis, we hypothesized that cartilage could be engineered by condensing chondrocytes in pellet culture and, in the present study, examined the quality of regenerated cartilage in direct comparison with native cartilage. Chondrocytes isolated from the sterna of chick embryos were cultured in pellets (4 × 10⁶ cells per pellet) for 2 weeks. Cartilage explants from the same source were cultured as controls. After 2 weeks, the regenerated cartilage from pellet culture had a disc shape and was on average 9 mm at the longest diameter. The chondrocyte phenotype was stabilized in pellet culture as shown by the synthesis of type II collagen and aggrecan, which was the same intensity as in the explant after 7 days in culture. During culture, chondrocytes also continuously synthesized type IX collagen. Type X collagen was negatively stained in both pellets and explants. Except for fibril orientation, collagen fibril diameter and density in the engineered cartilage were comparable with the native cartilage. In conclusion, hyaline cartilage engineered by chondrocytes in pellet culture, without the transformation of cell phenotypes and scaffold materials, shares similarities with native cartilage in cellular distribution, matrix composition and density, and ultrastructure.

Introduction

The lack of self-healing capacity in cartilage and the considerable morbidity caused by cartilage injuries and diseases have encouraged the search for a biomedical solution for repairing or restoration of damaged articular cartilage. The recent progress in tissue engineering of cartilage includes the introduction of new biomaterials for the scaffold, development of a bioreactor culture system, and the investigation of various cell resources and use of growth factors (Cao et al. 1998; Vunjak-Novakovic et al. 1999; Potter et al. 2000; Temenoff & Mikos, 2000; Solchaga et al. 2001; Cancedda et al. 2003). However, at the present time engineered cartilage still does not satisfy the need for functional cartilage repair (Anderer & Libera, 2002).

The phenotypic instability of chondrocytes in conventional monolayer culture has been a great challenge to cartilage engineering. In monolayer culture, chondrocytes typically devolve to a fibroblastic morphology and secrete type I collagen into the matrix but lose the expression of type II collagen and aggrecan core protein (Schnabel et al. 2002). The dedifferentiated chondrocytes reverse their phenotype when condensed by continuous culture after reaching confluence (Schulze-Tanzil et al. 2002). However, it is not clear to what extent the original chondrocyte phenotype is recapitulated during the redifferentiation process (Schnabel et al. 2002). In order to produce functional cartilage, it is crucial to avoid chondrocyte dedifferentiation during the process of cartilage engineering.

Cell density is one of the critical requirements for stabilizing the chondrocyte phenotype. When chondrocytes are plated at a high density, e.g. 4 × 10⁵ cm⁻² in culture flasks, their phenotypes do not change (Ruggiero et al. 1993). Similarly, chondrocyte pellet culture has provided an in vitro model of cartilage mineralization, usually involving growth plate chondrocytes (Kato et al. 1988; Farquharson & Whitehead, 1995). There is a very great distinction in the fate of chondrocytes between the growth plate, which is progressing towards calcification, and hyaline cartilage, such as articular cartilage, which normally does not calcify (Pacifici et al. 2000). Although some studies have explored the use of pellet culture for growth plate chondrocytes and the process of mineralization, few studies have examined hyaline cartilage chondrocytes in pellet culture. Published papers using pellet culture for hyaline cartilage chondrocytes have studied the effects of exogenous agents such as bone morphogenetic protein on the chondrocyte phenotype (Stewart et al. 2000), properties of extracellular matrix (Larson et al. 2002) and bioenergetics of chondrocytes (Croucher et al. 2000; Graff et al. 2000). Presently, no previous work has used this method to engineer hyaline cartilage, and subsequently evaluated the characteristics of neocartilage. The current study, utilizing chondrocytes from a hyaline cartilage source, was designed to engineer cartilage by pellet culture. The rationale behind this method is: (1) that chondrocyte phenotype is stabilized in the pellet, thereby avoiding dedifferentiation–redifferentiation; and (2) that cartilage is engineered without additional materials such as supporting scaffolds or gels (Vunjak-Novakovic et al. 1999; Temenoff & Mikos, 2000), which introduce possible complications including immune/inflammatory responses (Cancedda et al. 2003).

The chick sternum has been widely used in chondrocyte and cartilage studies because of its easy access and unique characteristics of development (Gibson & Flint, 1985; Hirsch & Svoboda, 1998; Liu et al. 1999;Tew et al. 2000; Zhang et al. 2002). The proximal sternum calcifies between gestational days 16 and 17, preceded by chondrocyte hypertrophy. In contrast, the distal sternum maintains a hyaline phenotype and does not calcify during development (Gibson & Flint, 1985; Craig et al. 1987). Thus, only the distal part of the chick sternum was utilized in the present study. In order to be functional, the regenerated cartilage is required to be structurally and biochemically identical to native cartilage (Caplan et al. 1997). Native cartilage explants served as control in the present study. In this study, the chondrocyte phenotype was assessed with immunohistochemistry of types I, II, IX, X collagen and aggrecan. Matrix deposition and the formation of a fibril network at the ultrastrucutural level were compared between the engineered cartilage and cartilage explants taken from the same source.

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Materials and methods

Pellet and explant cultures

Fertilized White Leghorn chicken eggs (Truslow Farms, Inc., Chestertown, MD, USA) were incubated at 37 °C for 16 days. The distal part of the sternum was removed from chick embryos, predigested in 0.2% collagenase (Worthington Biochemical Corp., Lakewood, NJ, USA) for 30 min, and further digested in fresh collagenase solution for 3 h. Chondrocytes were suspended in supplemented Dulbecco’s modified Eagle medium (DMEM; Biofluids Inc., Rockville, MD, USA) at a concentration of 10⁷ mL⁻¹. Four × 10⁶chondrocytes in 0.4 mL of cell suspension were transferred into each 0.75-mL tube (Matrix Technologies Corp., Lowell, MA, USA). Chondrocyte pellets were formed by centrifugation at 500 g for 10 min. The culture medium was DMEM plus 10% fetal bovine serum (Hyclone, Logan, UT, USA), 50 µg mL⁻¹ascorbate (Sigma, St Louis, MO, USA), 2 mm glutamine (Biofluids Inc.) and 0.2% penicillin/streptomycin (Life Technologies, Rockville, MD, USA). The pellets were cultured at 37 °C under a gas mixture of 95% air/5% CO₂. The pellets were transferred into Petri dishes at day 3 and continued to grow for 2 weeks. The medium was changed every day for the first 3 days and every second day for the rest of the culture period. Cell viability, regularly monitored with the LIVE/DEAD® kit (Molecular Probes, Eugene, OR, USA), was greater than 90% throughout the culture period.

For explant cultures, the distal parts of sterna were diced up to about 1 × 1 × 1 mm in size. One piece of the cartilage in 0.4 mL DMEM was added into the 0.75-mL tube and centrifuged in parallel to the chondrocytes at the time of pelletting. The explants were cultured in the tubes before being moved into Petri dishes at day 3. The culture conditions and medium were identical to pellet culture.

Samples were taken at days 1, 3, 7 and 14 and fixed in 4% paraformaldehyde for 1 h. They were then washed in phosphate-buffered saline (PBS) and passed through series of sucrose gradients at concentrations of 10%, 25% and 50%, and finally embedded in OCT compound (Sakura Finetechnical Co. Ltd, Tokyo, Japan) and sectioned at 5 µm with a cryostat for histology study. Samples for electron microscopy were collected on the same time schedule as histology but fixed separately (see below).

Immunohistochemistry

The primary antibodies for immunohistochemistry were mouse anti-avian collagens type I (SP1.D8), II (II-II6B3), IX (2C2) and X (X-AC9), and aggrecan link protein (9/30/8-A-4), obtained from the Developmental Studies Hybridoma Bank, The University of Iowa (Iowa City, IA, USA). Sections were predigested with 2 mg mL⁻¹ hyaluronidase (Sigma) for 30 min, or 1 h for type IX collagen staining (Vilamitjana et al. 1989), for the optimal penetration of antibodies. Before applying 9/30/8-A-4, aggrecan was reduced and alkylated by incubation with dithiothreitol and iodoacetic acid. After blocking with normal rabbit serum, primary antibody diluted in 1% bovine serum albumin (SP1.D8 1 : 10; II-II6B3, 2C2, X-AC9 and 9/30/8-A-4 1 : 5) was added onto the sections and incubated overnight at 4 °C. The incubation for Cy3-conjugated secondary antibody (Biomeda Biotechology, Foster City, CA, USA) was 30 min at room temperature. Sections were counterstained with 4′,6-diamidino-2-phenylindole (DAPI; Vector Laboratories, Burlingame, CA, USA). Primary antibody was replaced with PBS in the staining controls. Slides were viewed under an epifluorescent microscope and images were captured with a digital camera (Olympus BX51, Olympus America, Melville, NY, USA).

Histomorphometry

Histomorphometric analysis of aggrecan staining was performed on ten randomly selected sections of pellets and explants taken at scheduled time points, using Meta Imaging Series 4.6 (Universal Imaging Corp., West Chester, PA, USA). On each section, the measured areas, a computer-defined area of 350 × 350 pixels, were allocated as shown in Fig. 1. The average staining intensity in grey scale and the number of cells in the selected areas were calculated.

Fig. 1

Diagram of histomorphometry. On the pellet section, line c divides the section into two parts. Line b is the middle line of each part. The measuring area is on the medial side of line b. On the explant section, lines a and b are the intersect lines on …

Electron microscopy

Cartilage samples were processed as previously described (McCaffery & Farquhar, 1995). Briefly, samples for electron microscopy were fixed in fixative with 3.0% formaldehyde, 1.5% glutaraldehyde in 0.1 msodium cacodylate and 2.5% sucrose (pH 7.4) for 1 h at room temperature. They were then washed three times in 0.1 m sodium cacodylate/2.5% sucrose and post-fixed at 4 °C in Palade’s fixative containing 1% OsO₄. The samples were then washed, stained with uranyl acetate, dehydrated through a graded series of ethanol, and embedded in Epon. Eighty-nanometre sections were cut on a LEICA UCT ultramicrotome, post-stained in lead citrate and 2% uranyl acetate, and analysed on a Philips 420 TEM electron microscope (Royal Philips Electronics, The Netherlands) operated at 80 kV. The images were recorded and analysed with a Soft Imaging System Megaview III digital camera/software (Soft Imaging System Corp., Lakewood, CO, USA). Measurements of fibril diameter and the percentage of fibres were determined in 15 randomly selected views for the 2-week samples.

Statistical analysis

Data are presented as mean ± standard deviation and evaluated with Student’s t-test to compare the pellets with the explants at the same time point. Significance was defined as P < 0.05>

Results

The initial chondrocyte pellets, about 3.0 mm in diameter, at the bottom of the 0.75-mL tube became solid at day 3 when they were transferred from culture tubes into Petri dishes. The disc-like regenerated cartilage was about 9.0 mm in diameter and 1.0 mm in thickness by 2 weeks of culture (Fig. 2). When handling the samples for processing, the neocartilage showed similar rigidity and compression with the explant cartilage. The explants also grew and increased in size remarkably during the period of culture.

Fig. 2

Cartilage tissue generated by chondrocytes in pellet culture for 14 days. The initial number of chondrocytes was 4 × 10⁶ per pellet. The disc-shape tissue was hyaline in appearance (scale bar = 10 mm).

Immunohistochemistry and histomorphometry

Chondrocytes in both pellets and explants were round through the culture period. At day 1, chondrocyte density in the pellets (110 ± 17.9) was more than twice that in the explants (51 ± 9.5; P < 0.001). At day 3, a notable change in the pellets was the expansion of the intercellular space, accompanied by a gradual decrease in cell density in the pellets (80 ± 5). At day 7, cell density in the pellets (74 ± 8.5) was largely the same as in the explants (66 ± 8.4; P > 0.05), owing to the decreasing trend in the pellets and a peak increase in the explants. By day 14 cell density in the pellets (42 ± 7.8) and explants (37 ± 5.5; P > 0.05) was virtually identical.

Immunohistochemistry for aggrecan and types II and IX collagen demonstrated dynamic changes in matrix composition in both pellets and explants. Aggrecan was not seen around chondrocytes in the pellets until day 3 (Fig. 3). At day 3, the extracellular matrix in the pellets stained positively for aggrecan. Aggrecan in the pellets was mainly pericellularly stained, whereas it was uniformly stained throughout the extracellular space in the explants. The average intensity of aggrecan staining in the pellets was less than half that in the explants (Fig. 4). The average intensity of aggrecan staining in the explants steadily increased during the culture period. By day 7, aggrecan staining in the pellets showed the same pattern as in the explants and the average intensity in the pellets was equivalent to that in the explants (P > 0.05). At day 14, pericellular staining of aggrecan was the dominant pattern in both pellets and explants, and the average intensity was nearly identical.

Fig. 3

Immunohistochemistry for aggrecan on pellets (left column) and explants (right column). Reading from top to bottom, the rows reflect results at days 1, 3, 7 and 14. There was no aggrecan staining in the pellets, but was positively stained in the explants …

Fig. 4

The average staining intensity of aggrecan in the pellets and explants. Between days 1 and 3, aggrecan intensity in the pellets was significantly less then that in the explants. However, it was above the level of explants (P > 0.05) at day 7 and …

At day 1, type II collagen was not stained around chondrocytes in the pellets, whereas the extracellular matrix in explants was uniformly positive for type II collagen staining (Fig. 5). In the pellets, type II collagen staining was seen in the extracellular space at day 3 and was consistently positive up to day 14. In comparison with the explants, type II collagen was not evenly stained at day 7, and was more pericellularly distributed at day 14.

Fig. 5

Immunohistochemistry for type II collagen on pellets (left column) and explants (right column). Reading from top to bottom, the rows reflect results at days 1, 3, 7 and 14. Type II collagen was not stained in the pellets until day 3. At days 7 and 14, …

At day 1, type IX collagen was identified in the pellets and in the explants, and showed a more diffusive staining in the extracellular matrix of the pellets as compared with the explants (Fig. 6). The staining pattern was reminiscent of type IX collagen in both pellets and explants by day 3. Type IX collagen stained more intensely in the pellets in general, although it was unevenly stained in the matrix in both explants and pellets. By day 14, staining for type IX collagen in the pellets and explants shared the same pattern of both intracellular and extracellular staining.

Fig. 6

Immunohistochemistry for types IX (a–h) and X collagen (i, j) on pellets (left column) and explants (right column). Reading from top to bottom, the rows reflect results at days 1, 3, 7 and 14. Type IX collagen was positive in both pellets and …

Type I collagen was only stained at the edge of some of the pellet and explant sections. Type X collagen was negative in both the pellet and explant, even at the end of culture (Fig. 6I,j).

Ultrastructure

At day 1, chondrocytes in the pellets were at a high concentration, and some were next to each other. However, most of the intercellular space was electron-translucent (Fig. 7a). Chondrocytes in the explants were surrounded by highly organized matrix fibrils (Fig. 7b). Chondrocytes in both pellets and explants were rounded in shape, containing conspicuous nuclei with prominent endoplasmic reticulum and Golgi apparatus indicative of healthy, actively growing cells. At day 3, deposition of granular and fibril matrix was observed in the vicinity of chondrocytes in the pellets. The fibrils and macromolecules were sparse and randomly orientated around chondrocytes. No fibril network such as in the explants was formed (Fig. 7c,d). At day 7, the number of collagen fibrils was significantly increased in the pellets and the fibrils were assembled into a network. However, the fibril meshwork was still rather loose compared with that in the explants (Fig. 7e,f). Rough endoplasmic reticulum of chondrocytes in the pellets was less prominent than at day 3. Chondrocytes in the explants, but not in the pellets, showed an increasing number of vacuoles in cytoplasm. At day 14, the average density of collagen fibrils was 0.197 ± 0.066 in the pellets and 0.253 ± 0.054 in the explants (P = 0.103). The average diameter of collagen fibrils was not significantly different (pellets: 22.11 ± 3.32 nm; explants: 23.93 ± 2.89 nm, P = 0.085). However, there was a striking difference between the pellets and explants in the manner of fibril organization and orientation (Fig. 7g,h). Whereas collagen fibrils in the explants knitted into a well-connected network, the fibrils in the pellets appeared disorganized and randomly orientated.

Fig. 7

Pellets and explants: electron microscopy. At day 1, diminished electron density was seen in the intercellular space in the pellets. Chondrocytes in both pellets and explants exhibited extensive rough endoplasmic reticulum and Golgi complex (a, pellet, …

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Discussion

In direct comparison with native cartilage, this study characterized neocartilage generated by chondrocytes deriving from hyaline cartilage in pellet culture. After 1–2 weeks in culture, the neocartilage shared similarities with native cartilage with regard to chondrocyte phenotype, matrix distribution and the ultrastructure of collagen fibrils.

It has been suggested that an important consideration in engineering functional cartilage is to follow the principles of embryonic chondrogenesis (Solchaga et al. 2001). A critical step in chondrogenesis is the condensation of mesenchymal cells, which initiates cell–cell communication and leads to the birth of chondrocytes at the early stage of embryonic development (DeLise et al. 2000). Chondrocytes in the current study were condensed from a cell concentration of 1.0 × 10⁷ mL⁻¹ to a semisolid pellet. Chondrocyte density in the pellets increased two-fold over that in the explants. In the pellet, the close spatial relationship of neighboring chondrocytes and limited diffusion of newly formed matrix offer a better environment for cell–cell and cell–matrix communication (Farquharson & Whitehead, 1995) and facilitate regulation of chondrocyte differentiation (Hickok et al. 1998).

Retention of chondrocyte phenotype by the pellet culture was confirmed from the highly comparable staining patterns of immunohistochemistry for type II collagen, aggrecan and type IX collagen in the pellets with those in the cartilage explants. Type II collagen and aggrecan are tissue-specific markers for regenerated cartilage (Stewart et al. 2000; Anderer & Libera, 2002; Schulze-Tanzil et al. 2002) and are essential in distinguishing between hyaline cartilage and fibrocartilage, which has been responsible previously for the failure to generate a durable cartilage repair (Nehrer et al. 1999; Hunziker, 2001). At the same time, type I collagen synthesis was almost negligible in both pellet and explant culture. This also supports our notion that pellet culture is generating hyaline cartilage and not fibrocartilage.

The in vitro environment induces a much broader range of changes in chondrocyte dedifferentiation than previously thought (Schnabel et al. 2002). Type IX collagen, one of the fibril-associated collagens, has a role in connecting type II collagen and aggrecan (Olsen, 1997). In one study, the expression of type IX collagen was lost in dedifferentiated chondrocytes after monolayer culture, and did not recover when chondrocytes redifferentiated in alginate beads and re-expressed type II collagen and proteoglycan (Zaucke et al. 2001). This makes type IX collagen a sensitive marker of the tissue culture-associated redifferentiation of chondrocytes. In pellets, type IX collagen was steadily expressed through the 14 days of culture. Even at day 1 of pellet culture, when type II collagen and aggrecan were not detectable by immunohistochemistry and electron microscopy around the isolated chondrocytes, type IX collagen was immunohistochemically detected. Except for the conventional type II collagen and aggrecan, it is imperative to introduce additional markers for chondrocyte phenotype, such as type IX collagen, to monitor the process of cartilage engineering.

Active matrix production by chondrocytes in the pellets is evident by the remarkably increased size of the neocartilage and, at the same time, a gradually decreased cell density in the pellets during the culture period. The aggrecan concentration determines cartilage compression characteristics, while collagen fibril density, orientation and cross-linking determine its tensile property (Hasler et al. 1999). Scattered matrix macromolecules were observed in the pellet cultures as early as day 3. Quantitative immunohistochemistry for aggrecan indicates that aggrecan deposition by chondrocytes in the pellets quickly reached the level of cartilage explants during the period from day 3 to day 7, and it was maintained at virtually same level as in the explants up to day 14. This involved a continuous process of reconstruction of the fibril network in the pellets, as revealed by electron microscopy. By 2 weeks, the fibril meshwork in pellets was comparable with that of the explant cartilage. Several lines of evidence suggest that collagen fibrils developing in the pellets are similar to those found in native cartilage. Fibril diameter was not statistically different between the pellets and explant cartilage. Fibril density in pellets appeared lower than in explants, but this difference was also not statistically significant. However, it should be pointed out that even at day 14 the fibril orientation in the pellets did not have the same pattern as in native cartilage. The inferiority of the fibril network organization in the pellet-generated cartilage may ultimately impact on its biomechanical properties. One possible reason for the difference between the fibril network of the pellets and explants could be that there is a lack of mechanical stimulation during pellet culture; such physical stimulation exists during embryonic chondrogenesis of the explant cartilage. Combining mechanical stimulation with pellet culture could improve the fibril network because chondrocytes adapt to mechanical forces and remodel the organization of collagen fibres accordingly (Hasler et al. 1999), and also accelerate matrix production (Zhang et al. 2002).

In the model of cartilage regeneration we have described, the collagen fibril network formed between days 3 and 7, at which time the average intensity of aggrecan in the pellets reached the level seen in explants. This observation suggests that this period may be the optimal time for modulation of cartilage formation. By day 14, chondrocyte maturation was shown in the neocartilage, as indicated by the increased cellular volume. Although the marker for chondrocyte hypertrophy, type X collagen, was lacking, chondrocytes may proceed to hypertrophy quickly at this stage (Hirsch et al. 1996). When to terminate the culture to avoid chondrocyte hypertrophy is an important issue to consider, which has not been widely discussed in the field of tissue engineering.

We note that although the current study provides data on embryonic chick cartilage, certain clinical cartilage repair protocols are likely to be based upon the growth of mature human chondrocytes. Further investigation is required to specify appropriate growth characteristics in this context.

Conclusion

Hyaline cartilage was engineered by pellet culture of chondrocytes modelled after mesenchymal cell condensation in chondrogenesis. The pellet culture system retains the chondrocyte phenotype, and the resultant cartilage is therefore produced without the transformation of cell phenotypes or the need for scaffolding materials. Further interventions, such as mechanical stimulation, may be necessary to optimize the organization of collagen fibril network in pellet culture-generated cartilage.

Acknowledgments

This study was funded by the NIA Intramural Research Program, National Institutes of Health. We thank Dr Greta M. Lee for her help in pellet culture.