Inducer for regeneration of bone and soft tissue, and method for making same and uses thereof

Abstract

An inducer is directed to the induction of in situ regeneration in regenerative medicine. The inducer including an extracellular matrix and/or a bone morphogenetic protein, can induce the regeneration of bone and soft tissues surrounding the bone such as muscle, blood vessel and skin at the residual tissues where trauma occurs. The amount of regenerated tissue is associated with the dose of the implanted inducer.

Claims

1. A method for bone regeneration in a subject having a severed digit or limb, consisting essentially of: i) preparing an extracellular matrix (ECM) gel, wherein the ECM gel is prepared by: lyophilizing ECM harvested from porcine urinary bladder tissue to obtain a lyophilized ECM powder; and dispersing the lyophilized ECM powder in a humid environment having a humidity greater than 85%, so that it gets damp and hydrated to form an ECM gel; and ii) administering an effective amount of the ECM gel as the only active ingredient to the subject in need thereof, wherein the ECM gel is implanted into a lesion of the severed digit or limb.

2. The method of claim 1, wherein the severed digit or limb has soft tissues remained.

3. The method of claim 1, wherein the severed digit or limb does not have soft tissues distal to the surface of the severed digit or limb, and wherein administering the ECM gel regenerates both of bone and soft tissues.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 shows a picture of a normal mouse toe (ventral side, i.e., palm side).

(2) The middle toe was not amputated, while the toes (index toe and ring toe) at both sides of the middle toe were amputated at the second toe ridges of the index and ring toes (as indicated by the arrow). The third toe ridge of the middle toe was flush with the second toe ridges of the index and ring toes (as indicated by the arrow), so the third toe ridge of the middle toe can be used as a reference coordinate for the regeneration length of index and ring toes. Circled numbers 1 and 2 indicate the amputation positions.

(3) FIGS. 2-1 and 2-2 show pictures of mouse middle phalange (P2) 35 days after amputation and a transparent specimen thereof.

(4) FIGS. 2-1 shows a picture of mouse middle phalange (P2).

(5) FIGS. 2-2 shows a picture of a transparent specimen.

(6) The index toe was implanted with an inducer and the right toe (index toe) was used as a control without inducer. The transparent specimen was used to observe the bone. The single arrow indicates the amputation position and the double arrow indicates the length of P2. The length of the phalange implanted with inducer was twice that of the control phalange. FIGS. 2-1 and 2-2 refer to the same sample, and the ECM gel was used alone as the inducer. In FIG. 2-1: circled number 1: amputation position of the toe implanted with the inducer; circled number 2, regenerated toe through inducer implantation after amputation; circled number 3, normal toe (middle toe) without damage; circled number 4, control toe without inducer implantation after the amputation; and circled number 5, amputation position of the control toe. In FIG. 2-2: circled number 1, amputation position of the toe implanted with the inducer; circled number 2, regenerated toe through inducer implantation after amputation; circled number 3, length of the regenerated P2 bone; circled number 4, normal toe (middle toe) without damage; circled number 5, control toe without inducer implantation after amputation; circled number 6, amputation position of the control toe; and circled number 7, length of P2 bone of the control toe.

(7) FIGS. 3-1, 3-2 and 3-3 show micro-CT imaging and transparent specimen images of over-regenerated mouse P2 (different samples).

(8) FIG. 3-1 shows a micro-CT image (50 days after amputation);

(9) FIG. 3-2 shows a transparent specimen image (34 days after amputation); and

(10) FIG. 3-3 shows another transparent specimen image (35 days after amputation).

(11) The length of the regenerated P2 phalange was substantially equal to that of P2 of the normal middle toe, and the thickness of the regenerated toe was equal to or greater than that of the normal middle toe. The normal middle toe of mouse was substantially equal to the middle finger of the human, where P2 is the longest. The above results indicated that the bone mass of the regenerated P2 was greater than that of the original P2, which showed excessive regeneration of the mouse toe. A single arrow indicates the amputation position and a double arrow indicates the length of the P2. In FIG. 3-1: circled number 1, amputation position of the toe implanted with the inducer; circled number 2, regenerated P2; circled number 3, P3 bone of the normal middle toe; and circled number 4, P2 bone of the normal middle toe. In FIG. 3-2, circled number 1, length of the P2 bone of the normal middle toe; circled number 2, length of the regenerated P2 bone; and circled number 3, amputation position of the toe implanted with the inducer. In FIG. 3-3, circled number 1: amputation position of the toe implanted with the inducer; circled number 2, length of the P2 bone of the normal middle toe; and circled number 3, length of the regenerated P2 bone.

(12) FIG. 4 shows comparison of regenerative efficacies of the blank group, ECM alone group and BMP alone group. .diamond-solid. indicates p<0.05 for T test (p=0.011 for the ECM alone group vs. the blank group, and p=0.041 for the BMP alone group (500 μg/mL) vs. the blank group), .diamond-solid..diamond-solid. indicates p>0.05 for T test (p=0.908 for the ECM alone group vs. the BMP alone group).

(13) FIG. 5 shows comparison of regenerative efficacies of blank, ECM alone, combination of ECM and BMP for the amputated toe with soft tissues and the amputated toe without soft tissues. Combination-30 is a combination of ECM and 30 μg/mL of BMP, and the rest are named in the same way.

(14) FIG. 6 shows regenerative effect after amputation of the mouse tibia. The right picture shows a partial enlarged view of the left picture. In FIG. 6, number 1, amputation position of the mouse tibia (proximal end of ankle); number 2, a millimeter scale; and number 3, normal tibia.

DETAILED DESCRIPTION OF EMBODIMENTS

(15) The following embodiments are used to illustrate the invention in detail, but are not intended to limit the scope of the invention as defined by the appended claims.

Example 1

(16) 1. ECM was obtained from a fresh pig bladder and prepared into an ECM lyophilized powder according to a published method (Agrawal V, Johnson S A, Reing J. Epimorphic regeneration approach to tissue replacement in adult mammals [J]. Proc Natl Acad Sci, 2010, USA 107:3351-3355). The ECM lyophilized powder was dispersed in a humid environment with humidity greater than 85%, so that it was affected with damp and hydrated to prepare an ECM gel.

(17) 2. Recombinant human protein BMP-2 was purchased from Peprotech Inc. (Catalog #120-2). The BMP-2 was diluted to 100 μg/mL with a diluent to prepare a BMP-2 solution. Diluent for BMP-2 was a phosphate buffer (pH=7.2) containing 5% of trehalose.

(18) 3. Use methods of an amputated toe model and an inducer:

(19) Toes at both sides of a hind limb of a mouse were amputated at the distal second toe cross striation (middle of P2 bone), so that all tissues distal to the amputation surface were separated and removed. The inducer prepared as the above method was implanted into a wound to expose to the damaged bone tissue.

(20) 4. Method and result

(21) The ECM gel and the BMP-2 solution were mixed in an appropriate weight ratio to produce a mixture. The mixture was modeled and implanted into the stump of the mouse toe amputated at P2. In this example, the ECM gel was used at a dose of 0.25 mg/per toe (dry weight) and the BMP solution was used at amount of 0.25 μL/per toe, where a dry weight ratio of the BMP to the ECM was 1:10,000. After 35 days, length of the regenerated P2 was greater than that of the original P2; thickness of the regenerated P2 was equivalent to that of the original P2; and the bone mass of the regenerated P2 was more than that of the original P2, thereby showing excessive regeneration of the mouse digit. The results were shown FIGS. 1-3.

Example 2

(22) 2.1 Measurement (Bone Rate) of Regenerated Amount by the Inducer

(23) The mouse P2 bone amputation experiment showed that in the case where the amputated original tissues were removed, the inducer could induce synchronous regeneration of bone and soft tissues such as muscle and skin at a stump of the amputated toe, thereby increasing the length and the thickness of the amputated toe. The regenerated bone tissue had a pore structure and an uneven surface. Over time, the pore structure in the regenerated bone was reduced, and the bone surface tended to be smooth.

(24) Bone regeneration was a sign to determine whether regeneration occurred at the amputated toe, therefore, the regeneration effect used herein was expressed by relative bone mass, which was temporarily called “bone ratio” (see formula 1). In formula 1, the non-damaged toe was the middle toe, which corresponded to the middle finger of human, and the regenerated toes were toes at both sides of and adjacent to the middle toe, which corresponded to the index finger and the ring finger of human. The amputation position of the mouse toe was the middle of P2 bone of the toes at both sides (the index toe and ring toe), which corresponded to the second toe cross striation of the toes at both sides or the third toe cross striation of the middle toe of the body surface (see FIG. 1). The adult Kunming mouse without damage had a P2 bone ratio of 0.926 (i.e., the normal bone ratio was 0.926). The regeneration mass hereinafter was expressed by “bone rate”, which was a percentage obtained from the bone ratio through 0.926 (normal bone ratio)-normalization (see formula 2). For example, the normal bone rate is 100% in the case of no loss (=normal bone ratio÷0.926×100%).
bone ratio=P2 bone mass of the regenerated digit÷P2 bone mass of the digit without damage  Formula 1:
bone rate=bone ratio÷0.926×100%  Formula 2:

(25) (i.e., bone rate=P2 bone mass of the regenerated digit÷P2 bone mass of the digit without damage÷0.926×100%)

(26) 2.2 Regeneration of Amputated Toes Without Soft Tissues

(27) In this model, bones and soft tissues of the toes at both sides of the middle toe of on the mouse hind limbs were amputated at the middle of the P2 bone, so that all the tissues distal to the amputation surface were separated and removed. Four days after the amputation, when the wound was naturally closed, the inducer was implanted into the wound to expose to the residual section of the P2 bone. Thereafter, no treatment was performed until the mice were sacrificed 35 days after the implantation and the samples were collected for analysis. Used herein all were adult Kunming mice, each group of 3 mice, and 2 toes of the left and right limbs of each mouse were amputated, i.e., a total of 12 toes were amputated in each group.

(28) A blue gel bead group (Affi-Gel Blue Gel beads, purchased from Bio-Rad company, Hercules, Calif.), i.e., BMP alone group, was designed to compare the regenerative effects of ECM and BMPs according to a literature (Yu L, Han M, Yan M, et al. BMP2 induces segment-specific skeletal regeneration from digit and limb amputations by establishing a new endochondral ossification center. [J]. Developmental Biology. 2012, 372: 263-273). Concentration of BMP was 500 μg/mL, and diameter of the selected bead was 0.4 mm, so that more than 16.7 ng of the BMP can be carried (administration amount of the BMP in the combination-125 group was 15.6 ng/per toe). The ECM alone group indicated the absence of the BMP, where ECM was used at amount of 0.25 mg/per toe (dry weight). A group where toes at both sides were not implanted with an inducer after the amputation was called the blank group. The average bone rates of the blank group, ECM alone group and BMP alone group were 51.7%, 60.0% and 62.5%, respectively (see FIG. 4). P values obtained through comparison of the ECM alone group and the blank group and comparison of the BMP alone group and the blank group by T test were both less than 0.05, while p value obtained through comparison of the ECM alone group and the BMP alone group was greater than 0.05. Compared to the blank group, the average bone rate of the ECM alone group and the BMP alone group increased by 8.3% and 10.8%, respectively. The above results indicated that the ECM alone and BMP alone both showed a significant regenerative effect for the amputated toe, and their effects were similar.

(29) Mixed inducers of ECM and BMP were classified into 30, 60, 125, 250 and 500 (μg/mL) based on the concentration of BMP. The ECM gel was used at an amount of 0.25 mg/per toe (dry weight) and the BMP solution was used at a dose of 0.125 μL/per toe. A group added with 125 μg/mL of the BMP was named combination-125 group, and the other groups were named in the same way. For the combination-125 group, a dry weight ratio of the BMP to the ECM was 3:50,000, and an average bone rate was 83.0%, which increased by 31.3% compared to the blank group. Such results showed a significant synergistic effect produced by the combination of ECM and BMP. The regenerated bone mass induced by the mixed inducer of the ECM and the BMP increased with the increase of the BMP concentration, where a bone rate of the combination-250 group was 115.1%, which showed excessive bone regeneration; and a bone rate of the combination-500 group was 177.3%, which was about 3 times that of the ECM alone group (see FIG. 5).

(30) 2.3 Regeneration of Amputated Toes with Soft Tissues

(31) In this model, the P2 bone was amputated at the middle, and the P2 bone distal to the amputation surface and all the P3 bone were removed, but soft tissues such as skin were remained to encapsulate the regeneration inducer which was simultaneously implanted. Thereafter, no treatment was performed until the mice were sacrificed 34 days after the implantation and the samples were collected for analysis. The experimental group, and the composition and amount of the inducer were the same as those in 2.2.

(32) A bone rate of the combination-30 group was 101.4%, which showed excessive bone regeneration. The regenerated bone mass of the trauma model with soft tissue also increased with the increase of BMP concentration, but the increase was not obvious compared to the model without soft tissue. In addition, between the adjacent concentrations, significant difference was only observed between the combination-250 and the combination-500 (p=0.023). Except for the combination-500, the bone rate of each group in this model was greater than that of the corresponding group in the model with soft tissue. The bone rate of the combination-500 was about 1.5 times that of the ECM alone group (see FIG. 5). Such results demonstrated that the presence of soft tissues could significantly promote the regeneration of the phalange and the increase in BMP concentration led to different increases in the regeneration amount in different trauma models.

Example 3

(33) 1. ECM was obtained from a fresh pig small intestine, and the submucosa was separated by blunt machinery and then used to prepare an ECM lyophilized powder according to a reported method (Agrawal V, Johnson S A, Reing J. Epimorphic regeneration approach to tissue replacement in adult mammals. [J]. Proc Natl Acad Sci, 2010, USA 107:3351-3355).

(34) 2. Recombinant human protein BMP-9 was purchased from Peprotech company (Catalog #120-07). BMP-9 was diluted to a concentration of 60 μg/mL with a diluent, and the diluent was a phosphate buffer (pH=7.2) containing 5% of trehalose. After BMP-9 was completely dissolved, hydroxyapatite micropowder was introduced to the diluted BMP-9 solution, where the hydroxyapatite micropowder was purchased from Ding'an Technology Co., Ltd. (Suzhou).

(35) 3. The use methods of the amputation model and the inducer were described as follows. One hind limb bone of a mouse was amputated at a proximal end of the ankle (see FIG. 6) to separate and remove all the bone tissues distal to the amputation surface. Part of the soft tissue was retained so that the soft tissue section was located approximately 3 mm distal to the bone section. After the natural hemostasis, the inducer was implanted into the remained soft tissue to expose to the damaged bone tissue and allow for the natural wound closure. Thereafter, no treatment was performed.

(36) 4. The method and results were described below. The ECM lyophilized powder was mixed with the diluted BMP-9 solution in an appropriate weight ratio, where a dry weight ratio of BMP to ECM was 3:100,000. In this example, the amount of the ECM lyophilized powder was 4 mg/per limb; the amount of the BMP solution was 2 μL/per limb; and the amount of the hydroxyapatite micropowder was 0.2 mg/per limb. After 45 days, the samples were collected for analysis, and the results showed that the regenerated bone was tightly bonded to the original bone, and the regenerated bone had a size of 3.98×1.86×3.04 mm (FIG. 6).