Non-Thermal plasma is plasma that is not in thermodynamic equilibrium.  Atmospheric pressure plasma matches the pressure of the surrounding atmosphere.

Non-Thermal Atmospheric Pressure Plasma Enhances Mouse Limb Bud Survival, Growth and Elongation.

“appendage regeneration is dependent on reactive oxygen species (ROS) production and signaling. Mesenchymal cell stimulation by non-thermal (NT)-plasma, which produces and induces ROS, would (1) promote skeletal cell differentiation and (2) limb autopod development. Stimulation with a single treatment of NT-plasma enhanced survival, growth and elongation of mouse limb autopods in an in vitro organ culture system. Noticeable changes included enhanced development of digit length and definition of digit separation. These changes were coordinate with enhanced Wnt signaling in the distal apical epidermal ridge (AER) and presumptive joint regions. Autopod development continued to advance up to 144 hr in culture, seemingly overcoming the negative culture environment normally observed in this in vitro system. Real time qPCR analysis confirmed the up-regulation of chondrogenic transcripts. Mechanistically, NT-plasma increased the number of ROS positive cells in the dorsal epithelium, mesenchyme and the distal tip of each phalange behind the AER, determined using dihydrorhodamine. The importance of ROS production/signaling during development was further demonstrated by the stunting of digital outgrowth when anti-oxidants were applied. NT-plasma initiated and amplified ROS intracellular signaling to enhance development of the autopod. Parallels between development and regeneration, suggest the potential use of NT-plasma could extend to both tissue engineering and clinical applications to enhance fracture healing, trauma repair and bone fusion.”

“Vertebrate limbs grow through the coordinated activity of numerous growth factor signaling pathways, including the Wnt, fibroblast growth factor (FGF), bone morphogenetic protein (BMP), Notch, and transforming growth factor-β pathways. ROS play a primary role in mediating the activity of these factors and/or are integrally involved in regulating downstream signaling pathways. For example, a sustained increase in ROS levels is required for Wnt β-catenin signaling and the activation of one of its main downstream targets, fgf20. In addition, naturally occurring oxidative spikes are observed in most of the transition stages throughout developmental and regenerative processes. Furthermore, there is precedence for ROS production to both direct and enhance mesenchymal cell differentiation into either chondrocyte or osteoblast lineages”

“Since the NT-plasma discharge occurs in air, it is a highly complex mix of reactive chemical species (i.e., nitric oxide [NO], ROS, oxygen singles, and ozone), free ions, visible, ultraviolet, and near-infrared light, electric fields, and electromagnetic (EM) radiation.”

“one study showed that a 2 h treatment with EM field stimulated limb growth through increased proliferation and chondrocyte differentiation”

That is an absolutely huge elongation.

“Morphological formation of the joint region and alcian blue staining of proteoglycan appeared normal in sham and NT-plasma-treated digits. Chondrocytes in the proximal segment of the sham-treated digit were entering the prehypertrophic stage, based on the larger size, greater cellular spacing, and decrease in proteoglycan (white arrowhead). This typically precedes the formation of the primary center of ossification in the diaphysis. In contrast, chondrocytes from the NT-plasma-treated digit were immature and contained large amounts of proteoglycan. Even more striking was the much larger cartilage phalangeal segments in the NT-plasma-treated digit. A white line with double arrows marks the joint between the first and second phalanges in both sections. In the sham-treated digit, the adjacent joint spaces (white arrow) can be clearly observed; whereas in the NT-plasma-treated digits, there is no evidence of the next joint space, due to the increased segment length.”<-So maybe the plasma treatment increases stem cell differentiation to chondrocytes which is what we’d be looking for.

“Histology and quantitative real time-polymerase chain reaction were used to assess chondrogenesis and molecular events associated with the accelerated development. (A) A representative, hematoxylin and eosin (H&E) and alcian blue stained sections of middle phalanges from sham and NT-plasma-treated limb autopods. A white double arrow line marks the first joint space. The white arrows mark adjacent joint spaces, and the white arrowhead marks prehypertrophic chondrocytes in the sham control. (B) At 24 h post-NT-plasma treatment, alkaline phosphate (ALKP), catalase (CAT), endothelial nitric oxide synthase (eNOS), and bone morphogenetic protein 2 (BMP2) expression increased twofold above control. BMP4 and runt-related transcription factor 2 (Runx2) increased 12-fold and 8-fold, respectively, and fibroblast growth factor (FGF)-2 showed no increase. One hundred forty-four hours later, increases for ALKP (6-fold), CAT (14-fold), and BMP2 (3.5-fold). eNOS remained increased twofold above control at 144 h. BMP4 and Runx2 showed a relatively consistent elevated expression at 24 and 144 h. The results are expressed as the mean±standard deviation (n=3 [two limbs pooled per sample], *p<0.05). ”

“NT-plasma treatment promoted both growth and differentiation of cartilaginous elements within the embryonic mouse autopod. Based on the lack of limb growth after NT-plasma treatment in an antioxidant environment, and the increase in H2O2-positive cells in and under the epithelia throughout the 6 day culture period, we propose that NT-plasma induction of intracellular ROS also plays a role in the growth and differentiation observed in the autopod. In addition, H2O2-positive cells were observed in the distal digit tips behind the AER, concurrent with enhanced Wnt signaling. These findings are consistent with the ROS dependence of signaling factors (e.g., Wnt, BMP, and FGF) required for autopod development.”

“ROS production may not be the sole mechanism driving enhanced autopod growth. A second possible reason that NT-plasma enhanced autopod growth was its inhibition of epithelial overgrowth. ”

“In limb regeneration, a thickening of the epidermis has been shown to inhibit signaling to the blastema, halting the regeneration process.”

Hard to determine causality as there are so many potential causal factors as there are so many confounding variables in plasma.

Mice growth plates do not go through the fusion stage but they do cease eventually in longitudinal bone growth due to growth plate dysfunction.  If we can reverse growth plate dysfunction in mice than perhaps we can do so in humans and get more height from the growth plate.

This study however does not discuss reversing elderly growth plate dysfunction but rather states that older mice are still capable of undergoing endochondral ossification outside the growth plate.

Fractures in Geriatric Mice Show Decreased Callus Expansion and Bone Volume.

“Using a small animal model of long-bone fracture healing based on chronologic age, we asked how aging affected (1) the amount, density, and proportion of bone formed during healing; (2) the amount of cartilage produced and the progression to bone during healing; (3) the callus structure and timing of the fracture healing; and (4) the behavior of progenitor cells relative to the observed deficiencies of geriatric fracture healing.
Transverse, traumatic tibial diaphyseal fractures were created in 5-month-old (young adult) and 25-month-old (which we defined as geriatric, and are approximately equivalent to 70-85 year-old humans) C57BL/6 mice. Fracture calluses were harvested at seven times from 0 to 40 days postfracture for micro-CT analysis (total volume, bone volume, bone volume fraction, connectivity density, structure model index, trabecular number, trabecular thickness, trabecular spacing, total mineral content, bone mineral content, tissue mineral density, bone mineral density, degree of anisotropy, and polar moment of inertia), histomorphometry (total callus area, cartilage area, percent of cartilage, hypertrophic cartilage area, percent of hypertrophic cartilage area, bone and osteoid area, percent of bone and osteoid area), and gene expression quantification (fold change).
The geriatric mice produced a less robust healing response characterized by a pronounced decrease in callus amount (mean total volume at 20 days postfracture, 30.08 ± 11.53 mm3 versus 43.19 ± 18.39 mm3; p = 0.009), density (mean bone mineral density at 20 days postfracture, 171.14 ± 64.20 mg hydroxyapatite [HA]/cm3 versus 210.79 ± 37.60 mg HA/cm3; p = 0.016), and less total cartilage (mean cartilage area at 10 days postfracture, 101,279 ± 46,755 square pixels versus 302,167 ± 137,806 square pixels; p = 0.013) and bone content (mean bone volume at 20 days postfracture, 11.68 ± 3.18 mm3 versus 22.34 ± 10.59 mm3) compared with the young adult mice. However, the amount of cartilage and bone relative to the total callus size was similar between the adult and geriatric mice (mean bone volume fraction at 25 days postfracture, 0.48 ± 0.10 versus 0.50 ± 0.13), and the relative expression of chondrogenic (mean fold change in SOX9 at 10 days postfracture, 135 + 25 versus 90 ± 52) and osteogenic genes (mean fold change in osterix at 20 days postfracture, 22.2 ± 5.3 versus 18.7 ± 5.2; p = 0.324) was similar{so the deficiencies of older mice to undergo endochondral ossification in response to fracture may be related to things other than gene expression}. Analysis of mesenchymal cell proliferation in the geriatric mice relative to adult mice showed a decrease in proliferation (mean percent of undifferentiated mesenchymal cells staining proliferating cell nuclear antigen [PCNA] positive at 10 days postfracture, 25% ± 6.8% versus 42% ± 14.5%.
the molecular program of fracture healing is intact in geriatric mice, as it is in geriatric humans, but callus expansion is reduced in magnitude.
Our study showed altered healing capacity in a relevant animal model of geriatric fracture healing. The understanding that callus expansion and bone volume are decreased with aging can help guide the development of targeted therapeutics for these difficult to heal fractures.”

So older mice are still capable of chondrogenic fracture healing which is a lot like endochondral ossification.  Therefore, this provides evidence that older humans may be capable of inducing new endochondral ossification.

“At 10 days postfracture, corresponding to peak cartilage, young adult mice produced more total cartilage than geriatric mice (302,167 ± 137,806 versus 101,279 ± 46,755 square pixels”

“No difference was found in the percent cartilage content of the callus at 10 days postfracture (41.17% ± 10.13% versus 28.94% ± 8.74%) and in the percent of total cartilage that was hypertrophic at 15 days postfracture (88.76% ± 14.56% versus 70.21 ± 16.05%; p = 0.078). Osseous tissue formation did not reach high levels until 20 days postfracture with cartilage resorption nearly complete for both groups. Geriatric mice exhibit decreased total cartilage production and delayed resorption”

If you’re deficient in Cyp26b1 then having a Vitamin A deficient diet may be a way to grow taller.

Cyp26b1 Within the Growth Plate Regulates Bone Growth in Juvenile Mice.

“Retinoic acid (RA) is an active metabolite of vitamin A and plays important roles in embryonic development. CYP26 enzymes degrade RA and have specific expression patterns that produce a RA gradient, which regulates the patterning of various structures in the embryo.  Localized RA activities in the diaphyseal portion of the growth plate cartilage were associated with the specific expression of Cyp26b1 in the epiphyseal portion in juvenile mice. To disturb the distribution of RA, we generated mice lacking Cyp26b1 specifically in chondrocytes (Cyp26b1Δchon cKO). These mice showed reduced skeletal growth in the juvenile stage. Additionally, their growth plate cartilage showed decreased proliferation rates of proliferative chondrocytes, which was associated with a reduced height in the zone of proliferative chondrocytes, and closed focally by four weeks of age, while wild-type mouse growth plates never closed. Feeding the Cyp26b1 cKO mice a vitamin A-deficient diet partially reversed these abnormalities of the growth plate cartilage. Cyp26b1 in the growth plate regulates the proliferation rates of chondrocytes and is responsible for the normal function of the growth plate and growing bones in juvenile mice, probably by limiting the RA distribution in the growth plate proliferating zone.”

“Cyp26b1 is expressed in the distal region of developing limb buds, and mice that lack Cyp26b1 show severe limb malformation due to the spreading of the RA signal toward the distal end of the developing limb, causing abnormal patterning of limb skeletal elements”

“In juveniles, focal closures of the growth plate in the distal tibia, the proximal tibia, the distal tibia, elbow, proximal femur and distal femur are caused by the treatment of acne with retinoids or the treatment of hyperkeratinosis with cis-retinoic acid. In guinea pigs, the application of RA caused closure of the growth plates in the proximal tibia”

“Excess intake of vitamin A causes growth impairment and skeletal pain in juveniles”

Why Female Naked-Mole Rats and Meerkat Bones Grow Longer During Pregnancy

In the last month, I wrote the stunning post which suggested that a chemical that is associated with human reproduction might be the key to remodel and realign the human skeletal structure. Now, I show evidence that the phenomena of a female who is going through pregnancy is much more common that most people would believe.

So far, there has been two other vertebrates that researchers have studied and seen bone growth during pregnancy. They are the naked-mole rate and the meerkat. Tyler once asked me to look over the study on pregnancy of naked-mole rats and I decided to go back on those studies to look them over.

It seems that the reason these smaller mammal rodent-like creatures goes through body size increases is because they were kept from going through puberty.

As we might know, not all animal species has the same type of social structure as ours. When it comes to the naked-mole rats, they are what is known as eu-social.

There is just 1 female in an entire colony which can have babies, and somehow they can control the other females in the colony from ever going through puberty. It might be due to a certain chemical released or a completely social aspect. As the article states… “In most other mammals, puberty begins with the release of GnRH and activation of the hpg axis (Terasawa and Fernandez, 2001)”

The increases in the length of the lumbar spine region happens when the former subordinate females who could not go through puberty and become reproductively active is removed from the groups that they are in. Whatever was controlling them is removed.

The researchers not only removed the subordinate female out of the group, but also put them in a cage alone with a male, which caused in a few weeks vaginal perforation.

The result, even before the onset of pregnancy is that the female who is not no longer suppressed ends up much bigger in weight and length (by 82% and 37%, respectively).

Not only that, during pregnancy, the female also increases in length. If is only after she has given birth, and had not enough time to have pregnant again yet does the increase in lumbar length stop.

The researchers have now used this phenomena in the study to show that even the smaller, mammals seem to go through a phase similar to what we humans go through in puberty.

When we looked at the same type of phenomena happening the meerkat species, (Article: Adaptive Size Modification by Dominant Female Meerkats), we found that the bigger females and males were always the dominant one in the group, and would block reproductive chances by the other members of the group of the same sex. Only the most dominant and largest of the ones would mate and have litters.

When the researchers measured the levels of the hormones…

• Luteinizing Hormone (LH)
• Estradiol
• Cortisol

…In the females, they found that these hormones were raised in the dominant members.

The same was true when they looked at all the male members in the group and compared the…

• Testosterone
• Cortisol

….levels in the male and found the dominant one to have a higher level of the hormone.

I refer to three studies, “Cessation of Reproduction-Related Spine Elongation After Multiple Breeding Cycles in Female Naked Mole-Rats” and “Skeletal mass, chemistry, and growth during and after multiple reproductive cycles in the rat. Bone. [PubMed]” and “Growth and factors affecting body size in naked mole-rats.”

From the 1st study, the reason for the increase bone size is due to… “In other mammals (such as rats and humans), pregnancy-induced bone growth occurs to prepare the skeleton for the demands of lactation–a time of rapid bone depletion….While we did not measure bone loss in this study, it is possible that bone mineral resorption was occurring in naked mole-rats during the lactation period.“

It seems the the Meerkat subordinates suffer from the same type of endocrinology effect as what is seen in the subordinates in Naked-Mole Rats.

They lack both the circulating sex hormones & mature glands. This means that which they have all the right tools in their bodies, they are not triggered, until you give them a chance to be free from the suppression from the larger members of their groups.

This shows that the phenomena that we see in the smaller mammals is NOT another case of females pregnancy induced bone growth, but more of a puberty-like phase.

It seems that estrogen and progesterone, the chemical we already know effects human females during pregnancy, is also elevated in this rodent-like mammals during their pregnancy. However, that doesn’t help us much in our endeavor. Both of the hormones do contribute in bone formation and remodeling, but in us humans, almost all of us past a certain age have already gone through with puberty. There is a big difference in the reproductive patterns between humans and naked-mole rats and meerkats.

Click Here for more information and see the radiology pictures on how much a small subordinate female can change her body when she is allowed to grow and reproduce.