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.

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.

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.

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.

Copyright © 2011 Wiley Periodicals, Inc.

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

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.

Materials and methods

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.

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

• In addition, a complete list of the supplements is also available to the right of this home page. I have done as much research as I can to state with some confidence (but NOT completely confidence) that there is a chance that they will assist the body’s natural growing processes in either increasing the rate of growth or increase the ultimate height of an individual.

• You can see what are the types of PDFs, MP3s, Audio, Videos, etc I have managed to find from across the entire internet space which can possibly help you in the “- Free Stuff –” section. All of it is free and easy to download with a click of the button. Some of them I have paid for and others people have found for me.

Is there a question you wanted to ask?

• If you have any common questions, concerns, or issues, please go to the “Frequently Asked Questions” section and most of the most commonly raised questions and issues have already been answered to some degree. We are trying to consider all questions posed and have probably written up many answers to possible questions already in some way in the “Frequently Asked Question” section of the website.

• Please refer to there first before emailing us a message. We are trying to streamline the operations of the website so we can me more productive and effective in our output and work. If however you feel that you have a very pressing question or issue you need to get answered or resolved, you can email me using the email address at the top right side of the page.

To find out what resources are available right now to reference and utilize …

• It might be good to familiarize oneself with what tools, resources, and people one can get in touch with and use in this search. In the “Resources” section, there is a section which shows you a list of all of the posts that has ever been written on the website. In the list, you will find cool mind and body hacks which you can use and apply in your own life in case you would like to improve in other areas of life besides just height.

• In the “Podcast” section I plan to do a weekly or biweekly podcast where I talk about any topics which might be related to the endeavor of height increase, the science of auxology, medical pathologies, and other related subjects. I will try to find medical specialists and height increase experts but I would think it might be a very hard task to get them to be willing to go on the air and share their voice and opinions.

• Note: There is a “Forum” section to the website but that webpage is not activated at this time due to a personal judgement call made after discussing over the issue with other members of the community. Further considerations will be made in the future.

• In the “Scams List” section is a large collection of the names and products of products which are found on the internet which sell fake products or services which don’t work. Maybe there is a chance they might be effective for young children who are still growing but for the majority of the adults who are looking for a solution, they are just useless. Take a look at the lists and be informed on what is out there waiting to take your money.

I wish you all success in your height increase endeavors.

Sincerely,

Michael

Epilogue…

I have been getting a few emails recently which expressed their frustration and confusion over the fact that they can’t seem to be able to figure out what to do. They come to this website wanting to learn more about how to possibility increase in height and grow taller but the general problem is that this website is either too large, too complex, or too advanced in the science for them to understand what the going on.

I think I am finally realizing that I can’t do this entire project by myself. I thought that I could but it is far larger of a problem than expected. I need some help from collaborators, people who can step up and take action to push this thing further then just what I or the few others have done so far.

There is no way I can dedicate so much time to this project, as well as run my primary business company, as well as attend school, and still be able to accomplish other responsibilities. I also realized that if something happened to me (god forbid), there is no way another person can take up the task and continue on exactly where I left off. They would have to go back and redo all the research.

This is my way (and strategy) of distributing the work load out by making it easier for anyone who is really passionate and dedicated to the idea of height increase and want to quickly catch up on the necessary information and research so that they can make a contribution in research and possible make breakthroughs and innovation.

One of my biggest frustrations was trying to go back to find what my predecessors have already done, tried out, and proven ineffective so that I can know what to not put my energy towards and save some time in searching. Some of them kept good records but most ideas and techniques are randomly scattered across the internet space. I am going to try to make this easier for the people who come after me.